Testicular shape, scrotal skin thickness and testicular artery blood flow changes in bulls of different ages.

The aim of this study was to compare the biometric testicular characteristics, skin thickness and haemodynamics of the testicular artery of 12- and 24-month-old bulls using Doppler ultrasonography, the study was conducted using 48 indicus-taurus animals. The scrotal circumference (SC) and biometry characteristics of the bulls were measured to calculate the testicular volume. Doppler ultrasonography was used to obtain the haemodynamic values of the testicular artery. The skin thickness and volume were lower (p<.01) in the younger bulls (12 months:4.68 ± 0.68 mm; 168.76 ± 47.96 cm3 ) versus 24 months (5.05 ± 0.89; 499.73 ± 129.24 cm3 ) animals (p<.01). During diastole, mean velocity was lower in the 12 months (7.98 ± 3.83) than in the 24 months (11.37 ± 4.15) animals (p <.05). The 12-month-old animals had higher pulsatility and resistivity indices (0.49 ± 0.02; 0.51 ± 0.20) compared to the 24-month-old animals (0.32 ± 0.16; 0.40 ± 0.15) (p < .05). The final testicular end velocity was lower in animals with long/moderate-shaped (L/M) (7.31 ± 2.91) than in those moderate/oval-shaped (M/O) (11.48 ± 3.88) testicles (p < .05). Animals with L/M testes presented higher pulsatility values and resistivity indices (0.51 ± 0.05; 0.55 ± 0.04) compared to animals with M/O shape (0.29 ± 0.20; 0.36 ± 0.15). We showed that the blood flow of the supra testicular artery between the two evaluated ages differed, and that 24-month-old bulls presented better thermoregulation capacity. Animals with a long/moderate testicular format presented a greater vascular resistance, which was imposed on the blood flow due to the anatomical differences in the testicular artery, resulting in lower velocity, and indicating better heat dissipation in this format.

ANKHD1 is an S phase protein required for histone synthesis and DNA repair in multiple myeloma cells.

ANKHD1 is highly expressed in various cancers such as leukemia and multiple myeloma. Silencing of ANKHD1 expression leads to decreased cell proliferation and accumulation of cells at the S phase. In this study we found ANKHD1 expression to be higher at the S phase, suggesting it to be an S phase protein. We observed that ANKHD1 interacts with histone promoter regions and its inhibition downregulates expression of all core histones, implying a role in histone synthesis. Since histone synthesis occurs in parallel with DNA replication at S phase, we evaluated PCNA (Proliferating Cell Nuclear Antigen) expression, a protein involved in DNA replication and repair. PCNA expression was found to be significantly decreased in ANKHD1 silenced cells. We further observed accumulation γH2AX, a marker for DNA double stranded breaks and an early sign of DNA damage induced by replication stress, upon ANKHD1 silencing. The expressions of several genes implicated in DNA repair were also modulated in ANKHD1 silenced cells, confirming the role of ANKHD1 in DNA repair. Based on this study we speculate that ANKHD1 is an S phase protein required for histone synthesis and DNA repair. These results however, are preliminary and require thorough investigation.

Up-regulation of SPINT2/HAI-2 by Azacytidine in bone marrow mesenchymal stromal cells affects leukemic stem cell survival and adhesion.

The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz-type2 (SPINT2/HAI-2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI-2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5-Azacitidine (Aza), a DNA methyltransferase inhibitor. In MDS- and de novo AML-BMMSC, Aza treatment resulted in a pronounced SPINT2/HAI-2 levels up-regulation. Moreover, Aza treatment of HD-BMMSC did not improve SPINT2/HAI-2 levels. To understand the role of SPINT2/HAI-2 down-regulation in BMMSC physiology, SPINT2/HAI-2 expression was inhibited by lentivirus. SPINT2 underexpression resulted in an increased production of HGF by HS-5 stromal cells and improved survival of CD34+ de novo AML cells. We also observed an increased adhesion of de novo AML hematopoietic cells to SPINT2/HAI-2 silenced cells. Interestingly, BMMSC isolated from MDS and de novo AML patients had increased expression of the integrins CD49b, CD49d, and CD49e. Thus, SPINT2/HAI-2 may contribute to functional and morphological abnormalities of the microenvironment niche and to stem/progenitor cancer cell progression. Hence, down-regulation in SPINT2/HAI-2 gene expression, due to methylation in MDS-BMMSC and de novo AML-BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment.

Infrared thermography relationship between the temperature of the vulvar skin, ovarian activity, and pregnancy rates in Braford cows.

The objective of this study was to compare the presence of ovarian activity and pregnancy rates to temperature variation at the vulvar skin measured by infrared thermography (IRT). In addition, we also aimed to evaluate the IRT as a non-invasive method to evaluate animal breeding from fixed timed artificial insemination (FTAI). The study comprises 150 non-lactating beef Braford cows (5/8 Hereford × 3/8 Nellore) aged between 3 and 10 years. Data were collected along the FTAI protocol period during animal management. Animals were subjected to reproductive ultrasound evaluation and thermal images were performed by an infrared camera. Mean skin vulvar temperature (°C) and ovarian structures data were compared using Tukey's t test used as follow-up test to ANOVA. We observed a statistical difference in the mean vulvar skin temperature between animals that had the presence of ovarian follicles (34.2 ± 1.8) compared to no activity (35.4 ± 1.0; P < 0.05). However, vulvar skin temperature were similar between pregnant (34.5 ± 1.5) compared to non-pregnant (34.3 ± 1.9) animals (P > 0.05). In conclusion, the IRT technique was efficient to detect changes on vulvar skin temperature observed during FTAI protocol in Braford cows. Therefore, the use of IRT as an indirectly diagnostic tool to detect ovarian activity seems promising and further studies are required to validate their potential in beef cattle production.

ANKHD1 silencing inhibits Stathmin 1 activity, cell proliferation and migration of leukemia cells.

ANKHD1 is highly expressed in human acute leukemia cells and potentially regulates multiple cellular functions through its ankyrin-repeat domains. In order to identify interaction partners of the ANKHD1 protein and its role in leukemia cells, we performed a yeast two-hybrid system screen and identified SIVA, a cellular protein known to be involved in proapoptotic signaling pathways. The interaction between ANKHD1 and SIVA was confirmed by co-imunoprecipitation assays. Using human leukemia cell models and lentivirus-mediated shRNA approaches, we showed that ANKHD1 and SIVA proteins have opposing effects. While it is known that SIVA silencing promotes Stathmin 1 activation, increased cell migration and xenograft tumor growth, we showed that ANKHD1 silencing leads to Stathmin 1 inactivation, reduced cell migration and xenograft tumor growth, likely through the inhibition of SIVA/Stathmin 1 association. In addition, we observed that ANKHD1 knockdown decreases cell proliferation, without modulating apoptosis of leukemia cells, while SIVA has a proapoptotic function in U937 cells, but does not modulate proliferation in vitro. Results indicate that ANKHD1 binds to SIVA and has an important role in inducing leukemia cell proliferation and migration via the Stathmin 1 pathway. ANKHD1 may be an oncogene and participate in the leukemia cell phenotype.

Ten-eleven-translocation 2 (TET2) is downregulated in myelodysplastic syndromes.

TET2, a member of the ten-eleven-translocation (TET) family genes that modify DNA by converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), is located in chromosome 4q24 and is frequently mutated in myeloid malignancies. The impact of TET2 mutation on survival outcomes is still controversial; however, functional studies have proved that it is a loss-of-function mutation that impairs myeloid cell differentiation and contributes to the phenotype of myeloid neoplasia. We, herein, aimed to investigate TET2 expression in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A significantly decreased TET2 expression was observed in bone marrow cells from AML (n = 53) and patients with MDS (n = 64), compared to normal donors (n = 22). In MDS, TET2 expression was significantly reduced in RAEB-1/RAEB-2 compared to other WHO 2008 classifications, and a lower TET2 expression was observed at the time of MDS disease progression in four of five patients. In multivariate analysis, low TET2 expression (P = 0.03), male gender (P = 0.02), and WHO 2008 classification (P < 0.0001) were independent predictors of poorer overall survival. These results suggest that defective TET2 expression plays a role in the MDS pathophysiology and predicts survival outcomes in this disease.

ANKHD1, a novel component of the Hippo signaling pathway, promotes YAP1 activation and cell cycle progression in prostate cancer cells.

ANKHD1 is a multiple ankyrin repeat containing protein, recently identified as a novel member of the Hippo signaling pathway. The present study aimed to investigate the role of ANKHD1 in DU145 and LNCaP prostate cancer cells. ANKHD1 and YAP1 were found to be highly expressed in prostate cancer cells, and ANKHD1 silencing decreased cell growth, delayed cell cycle progression at the S phase, and reduced tumor xenograft growth. Moreover, ANKHD1 knockdown downregulated YAP1 expression and activation, and reduced the expression of CCNA2, a YAP1 target gene. These findings indicate that ANKHD1 is a positive regulator of YAP1 and promotes cell growth and cell cycle progression through Cyclin A upregulation.

An experimental universal swine influenza a virus (IAV) vaccine candidate based on the M2 ectodomain (M2e) peptide does not provide protection against H1N1 IAV challenge in pigs.

Swine flu is a common disease problem in North American pig populations and swine influenza A viruses (IAV) are extremely diverse and the lack of cross protection between heterologous strains is impacting vaccine efficacy in the field. The objective of this study was to design and test a novel swine flu vaccine targeting the M2 ectodomain (M2e) of IAV, a highly conserved region within the IAV proteome. In brief, an M2e peptide was designed to match the predominant swine IAV M2 sequence based on global analysis of sequences from pigs and humans. The resulting sequence was used to synthesize the M2e peptide coupled to a carrier protein. The final vaccine concentration was 200 µg per dose, and a commercial, microemulsion-based aqueous adjuvant was added. Nine 3-week-old IAV negative piglets were randomly assigned to three groups and rooms including non-vaccinated pigs (NEG-CONTROLs) and vaccinated pigs using the intramuscular (M2e-IM) or the intranasal route (M2e-IN). Vaccinations were done at weaning and again at 2 weeks later. An in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated to study the M2e IgG antibody response and demonstrated M2e-IM pigs had a higher systemic antibody response compared to M2e-IN pigs. Subsequently, an IAV challenge study was conducted. The results indicated that M2e-IM vaccinated pigs were not protected from H1N1 (US pandemic clade, global clade 1A.3.3.2) challenge despite having a strong humoral anti-M2e immune response. In conclusion, while the experimental IAV vaccine was able to induce anti-M2e antibodies, when challenged with H1N1, the vaccinated pigs were not protected, perhaps indicating that reactivity to the M2e antigen alone is not sufficient to reduce clinical signs, lesions or shedding associated with experimental IAV challenge.

Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors.

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34+ progenitors prior to B/T cell emergence. While these TdT+ cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis.

Autophagy counters inflammation-driven glycolytic impairment in aging hematopoietic stem cells.

Autophagy is central to the benefits of longevity signaling programs and to hematopoietic stem cell (HSC) response to nutrient stress. With age, a subset of HSCs increases autophagy flux and preserves regenerative capacity, but the signals triggering autophagy and maintaining the functionality of autophagy-activated old HSCs (oHSCs) remain unknown. Here, we demonstrate that autophagy is an adaptive cytoprotective response to chronic inflammation in the aging murine bone marrow (BM) niche. We find that inflammation impairs glucose uptake and suppresses glycolysis in oHSCs through Socs3-mediated inhibition of AKT/FoxO-dependent signaling, with inflammation-mediated autophagy engagement preserving functional quiescence by enabling metabolic adaptation to glycolytic impairment. Moreover, we show that transient autophagy induction via a short-term fasting/refeeding paradigm normalizes glycolytic flux and significantly boosts oHSC regenerative potential. Our results identify inflammation-driven glucose hypometabolism as a key driver of HSC dysfunction with age and establish autophagy as a targetable node to reset oHSC regenerative capacity.

Lower expression of NOTCH components in peripheral blood mononuclear cells of allogeneic hematopoietic cell transplant patients.

INTRODUCTION: Chronic graft-versus-host disease (cGvHD) not only remains the main cause of late mortality after allogeneic hematopoietic cell transplant, but also has the capacity of causing severe organ impairment in those who survive. The Notch, a highly conserved ligand-receptor pathway, is involved in many immunological processes, including inflammatory and regulatory responses. Recently, mouse models have shown that the blockage of canonical Notch signaling prevents GvHD. OBJECTIVE AND METHOD: Due to the lack of data on the Notch pathway in human chronic GvHD, we sought to study the expression of NOTCH components in primary samples of patients who received allo-HCT and presented active cGvHD or a long-term clinical tolerance to cGvHD. RESULTS: Our results showed a significantly lower expression of NOTCH components in both groups that received allo-HCT, independently of their cGvHD status, when compared to healthy controls. CONCLUSION: Moreover, there were no differences in gene expression levels between the active cGvHD and clinically tolerant groups. To our knowledge, this is one of the first studies performed in human primary samples and our data indicate that much remains to be learned regarding NOTCH signaling as a new regulator of GvHD.

Polyunsaturated Fatty Acid-Bound α-Fetoprotein Promotes Immune Suppression by Altering Human Dendritic Cell Metabolism.

α-Fetoprotein (AFP) is expressed by stem-like and poor outcome hepatocellular cancer tumors and is a clinical tumor biomarker. AFP has been demonstrated to inhibit dendritic cell (DC) differentiation and maturation and to block oxidative phosphorylation. To identify the critical metabolic pathways leading to human DC functional suppression, here, we used two recently described single-cell profiling methods, scMEP (single-cell metabolic profiling) and SCENITH (single-cell energetic metabolism by profiling translation inhibition). Glycolytic capacity and glucose dependence of DCs were significantly increased by tumor-derived, but not normal cord blood-derived, AFP, leading to increased glucose uptake and lactate secretion. Key molecules in the electron transport chain in particular were regulated by tumor-derived AFP. These metabolic changes occurred at mRNA and protein levels, with negative impact on DC stimulatory capacity. Tumor-derived AFP bound significantly more polyunsaturated fatty acids (PUFA) than cord blood-derived AFP. PUFAs bound to AFP increased metabolic skewing and promoted DC functional suppression. PUFAs inhibited DC differentiation in vitro, and ω-6 PUFAs conferred potent immunoregulation when bound to tumor-derived AFP. Together, these findings provide mechanistic insights into how AFP antagonizes the innate immune response to limit antitumor immunity. SIGNIFICANCE: α-Fetoprotein (AFP) is a secreted tumor protein and biomarker with impact on immunity. Fatty acid-bound AFP promotes immune suppression by skewing human dendritic cell metabolism toward glycolysis and reduced immune stimulation.

Unravelling human hematopoietic progenitor cell diversity through association with intrinsic regulatory factors.

Hematopoietic stem and progenitor cell (HSPC) transplantation is an essential therapy for hematological conditions, but finer definitions of human HSPC subsets with associated function could enable better tuning of grafts and more routine, lower-risk application. To deeply phenotype HSPCs, following a screen of 328 antigens, we quantified 41 surface proteins and functional regulators on millions of CD34+ and CD34- cells, spanning four primary human hematopoietic tissues: bone marrow, mobilized peripheral blood, cord blood, and fetal liver. We propose more granular definitions of HSPC subsets and provide new, detailed differentiation trajectories of erythroid and myeloid lineages. These aspects of our revised human hematopoietic model were validated with corresponding epigenetic analysis and in vitro clonal differentiation assays. Overall, we demonstrate the utility of using molecular regulators as surrogates for cellular identity and functional potential, providing a framework for description, prospective isolation, and cross-tissue comparison of HSPCs in humans.

Autophagy counters inflammation-driven glycolytic impairment in aging hematopoietic stem cells.

Aging of the hematopoietic system promotes various blood, immune and systemic disorders and is largely driven by hematopoietic stem cell (HSC) dysfunction ( 1 ). Autophagy is central for the benefits associated with activation of longevity signaling programs ( 2 ), and for HSC function and response to nutrient stress ( 3,4 ). With age, a subset of HSCs increases autophagy flux and preserves some regenerative capacity, while the rest fail to engage autophagy and become metabolically overactivated and dysfunctional ( 4 ). However, the signals that promote autophagy in old HSCs and the mechanisms responsible for the increased regenerative potential of autophagy-activated old HSCs remain unknown. Here, we demonstrate that autophagy activation is an adaptive survival response to chronic inflammation in the aging bone marrow (BM) niche ( 5 ). We find that inflammation impairs glucose metabolism and suppresses glycolysis in aged HSCs through Socs3-mediated impairment of AKT/FoxO-dependent signaling. In this context, we show that inflammation-mediated autophagy engagement preserves functional quiescence by enabling metabolic adaptation to glycolytic impairment. Moreover, we demonstrate that transient autophagy induction via a short-term fasting/refeeding paradigm normalizes glucose uptake and glycolytic flux and significantly improves old HSC regenerative potential. Our results identify inflammation-driven glucose hypometabolism as a key driver of HSC dysfunction with age and establish autophagy as a targetable node to reset old HSC glycolytic and regenerative capacity. One-Sentence Summary: Autophagy compensates for chronic inflammation-induced metabolic deregulation in old HSCs, and its transient modulation can reset old HSC glycolytic and regenerative capacity.

Genome-wide analysis of aberrant position and sequence of plasma DNA fragment ends in patients with cancer.

Genome-wide fragmentation patterns in cell-free DNA (cfDNA) in plasma are strongly influenced by cellular origin due to variation in chromatin accessibility across cell types. Such differences between healthy and cancer cells provide the opportunity for development of novel cancer diagnostics. Here, we investigated whether analysis of cfDNA fragment end positions and their surrounding DNA sequences reveals the presence of tumor-derived DNA in blood. We performed genome-wide analysis of cfDNA from 521 samples and analyzed sequencing data from an additional 2147 samples, including healthy individuals and patients with 11 different cancer types. We developed a metric based on genome-wide differences in fragment positioning, weighted by fragment length and GC content [information-weighted fraction of aberrant fragments (iwFAF)]. We observed that iwFAF strongly correlated with tumor fraction, was higher for DNA fragments carrying somatic mutations, and was higher within genomic regions affected by copy number amplifications. We also calculated sample-level means of nucleotide frequencies observed at genomic positions spanning fragment ends. Using a combination of iwFAF and nine nucleotide frequencies from three positions surrounding fragment ends, we developed a machine learning model to differentiate healthy individuals from patients with cancer. We observed an area under the receiver operative characteristic curve (AUC) of 0.91 for detection of cancer at any stage and an AUC of 0.87 for detection of stage I cancer. Our findings remained robust with as few as 1 million fragments analyzed per sample, demonstrating that analysis of fragment ends can become a cost-effective and accessible approach for cancer detection and monitoring.

Knockdown of insulin receptor substrate 1 reduces proliferation and downregulates Akt/mTOR and MAPK pathways in K562 cells.

BCR-ABL kinase activates downstream signaling pathways, including the PI3K-Akt/mTOR and the MAPK pathway. IRS1 has been previously described as constitutively phosphorylated and associated with BCR-ABL in K562 cells, suggesting that IRS1 has role in the BCR-ABL signaling pathways. In this study, we analyzed the effect of IRS1 silencing, by shRNA-lentiviral delivery, in K562 cells, a CML cell line that presents the BCR-ABL. IRS1 silencing decreased cell proliferation and colony formation in K562 cells, which correlates with the delay of these cells at the G0/G1 phase and a decrease in the S phase of the cell cycle. Furthermore, IRS1 silencing in K562 cells resulted in a decrease of Akt, P70S6K and ERK1/2 phosphorylation. Nevertheless, apoptosis was unaffected by IRS1 knockdown and no alterations were found in the phosphorylation of BAD and in the expression of BCL2 and BAX. BCR-ABL and CRKL phosphorylation levels remained unaffected upon IRS1 silencing, and no synergistic effect was observed with imatinib treatment and IRS1 knockdown, indicating that IRS1 is downstream from BCR-ABL. In conclusion, we demonstrated that inhibition of IRS1 is capable of inducing the downregulation of Akt/mTOR and MAPK pathways and further decreasing proliferation, and clonogenicity and induces to cell cycle delay at G0/G1 phase in BCR-ABL cells.

Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B.

Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

Expression of transforming growth factor ß pathway components in chronic graft-versus-host disease after allogeneic hematopoietic cell transplantation.

Chronic graft-versus-host disease (cGvHD), an immunological complication of allogeneic cell transplantation, is the principal cause of non-relapse mortality and morbidity. Even though advances have been made in understanding the pathophysiology of this disorder, many questions remain. We sought to evaluate gene expression of transforming growth factor ß (TGF-ß) pathway components, through quantitative RT-PCR and PCR array, in patients with cGvHD with different disease activity. We observed an upregulation of SMAD3, BMP2, CDKN1A, IL6, and TGF-ß2 genes in the clinical tolerance group, which had never developed cGvHD, or which had been withdrawn from all immunosuppressive treatments (IST) for at least 1 year. In addition, SMAD5 gene upregulation was observed in cGvHD patients undergoing IST, and ordinal regression showed a correlation between SMAD5 expression and disease severity. Our data support the evidence of the important role of TGF-ß effects in the pathological process of cGvHD.

Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment.

Master transcription factors (TFs) directly regulate present and future cell states by binding DNA regulatory elements and driving gene-expression programs. Their abundance influences epigenetic priming to different cell fates at the chromatin level, especially in the context of differentiation. In order to link TF protein abundance to changes in TF motif accessibility and open chromatin, we developed InTAC-seq, a method for simultaneous quantification of genome-wide chromatin accessibility and intracellular protein abundance in fixed cells. Our method produces high-quality data and is a cost-effective alternative to single-cell techniques. We showcase our method by purifying bone marrow (BM) progenitor cells based on GATA-1 protein levels and establish high GATA-1-expressing BM cells as both epigenetically and functionally similar to erythroid-committed progenitors.

Feasibility of circulating tumor DNA analysis in dogs with naturally occurring malignant and benign splenic lesions.

Comparative studies of naturally occurring canine cancers have provided new insight into many areas of cancer research. Development and validation of circulating tumor DNA (ctDNA) analysis in pet dogs can help address diagnostic needs in veterinary as well as human oncology. Dogs have high incidence of naturally occurring spontaneous cancers, demonstrate molecular heterogeneity and clonal evolution during therapy, allow serial sampling of blood from the same individuals during the course of disease progression, and have relatively compressed intervals for disease progression amenable to longitudinal studies. Here, we present a feasibility study of ctDNA analysis performed in 48 dogs including healthy dogs and dogs with either benign splenic lesions or malignant splenic tumors (hemangiosarcoma) using shallow whole genome sequencing (sWGS) of cell-free DNA. To enable detection and quantification of ctDNA using sWGS, we adapted two informatic approaches and compared their performance for the canine genome. At the time of initial clinical presentation, mean ctDNA fraction in dogs with malignant splenic tumors was 11.2%, significantly higher than dogs with benign lesions (3.2%; p = 0.001). ctDNA fraction was 14.3% and 9.0% in dogs with metastatic and localized disease, respectively (p = 0.227). In dogs treated with surgical resection of malignant tumors, mean ctDNA fraction decreased from 11.0% prior to resection to 7.9% post-resection (p = 0.047 for comparison of paired samples). Our results demonstrate that ctDNA analysis is feasible in dogs with hemangiosarcoma using a cost-effective approach such as sWGS. Additional studies are needed to validate these findings, and determine the role of ctDNA to assess burden of disease and treatment response in dogs with cancer.