Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis.

Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.

Superfluid Helium Drops Levitated in High Vacuum.

We demonstrate the trapping of millimeter-scale superfluid helium drops in high vacuum. The drops are sufficiently isolated that they remain trapped indefinitely, cool by evaporation to 330 mK, and exhibit mechanical damping that is limited by internal processes. The drops are also shown to host optical whispering gallery modes. The approach described here combines the advantages of multiple techniques, and should offer access to new experimental regimes of cold chemistry, superfluid physics, and optomechanics.

Cryogenic and hermetically sealed packaging of photonic chips for optomechanics.

We demonstrate a hermetically sealed packaging system for integrated photonic devices at cryogenic temperatures with plug-and-play functionality. This approach provides the ability to encapsulate a controlled amount of gas into the optical package allowing helium to be used as a heat-exchange gas to thermalize photonic devices, or condensed into a superfluid covering the device. This packaging system was tested using a silicon-on-insulator slot waveguide resonator which fills with superfluid 4He below the transition temperature. To optimize the fiber-to-chip optical integration 690 tests were performed by thermally cycling optical fibers bonded to various common photonic chip substrates (silicon, silicon oxide and HSQ) with a range of glues (NOA 61, NOA 68, NOA 88, NOA 86H and superglue). This showed that NOA 86H (a UV curing optical adhesive with a latent heat catalyst) provided the best performance under cryogenic conditions for all the substrates tested. The technique is relevant to superfluid optomechanics experiments, as well as quantum photonics and quantum optomechanics applications.

Carbohydrate mouth rinsing does not affect 6-min walk test performance and blood glucose responses in older adults.

PURPOSE: Carbohydrate (CHO) mouth rinsing (MR) prior to exercise has been shown to elicit enhanced performance and energy availability in some studies. Previous literature has concentrated on examining CHO MR strategies for improving aerobic endurance performance in younger athletic adults. Knowledge of the impact of CHO MR on functional performance in older adults is scarce. The purpose of this investigation was to determine if CHO MR would improve 6-min walk test (6MWT) performance, perceived exertion, and blood glucose responses in older adults. METHOD: Thirty-three individuals (16 males, 17 females), age ≥ 70 years performed two 6MWT trials, one of which utilized a 6.4% maltodextrin CHO MR and one of which utilized a placebo MR. Participants held the MR in their mouth for 20 s prior to the 6MWT, and trials occurred in a counterbalanced fashion. Total distance walked and rating of perceived exertion (RPE) were recorded upon completion of each 6MWT. Heart rate (HR), peripheral blood oxygen saturation (SpO2), systolic and diastolic blood pressures (BP), blood glucose, and blood lactate were measured before and after each 6MWT. RESULT: CHO MR did not alter the response of any study parameter compared to the placebo MR (p = 0.13-0.94). HR, systolic BP, and blood lactate increased and SpO2 decreased across time (p < 0.01). CONCLUSION: A 6.4% maltodextrin CHO MR did not alter total distance walked, perceived exertion, or other physiological responses elicited by the 6MWT in older adults.

DRUG-NEM: Optimizing drug combinations using single-cell perturbation response to account for intratumoral heterogeneity.

An individual malignant tumor is composed of a heterogeneous collection of single cells with distinct molecular and phenotypic features, a phenomenon termed intratumoral heterogeneity. Intratumoral heterogeneity poses challenges for cancer treatment, motivating the need for combination therapies. Single-cell technologies are now available to guide effective drug combinations by accounting for intratumoral heterogeneity through the analysis of the signaling perturbations of an individual tumor sample screened by a drug panel. In particular, Mass Cytometry Time-of-Flight (CyTOF) is a high-throughput single-cell technology that enables the simultaneous measurements of multiple ([Formula: see text]40) intracellular and surface markers at the level of single cells for hundreds of thousands of cells in a sample. We developed a computational framework, entitled Drug Nested Effects Models (DRUG-NEM), to analyze CyTOF single-drug perturbation data for the purpose of individualizing drug combinations. DRUG-NEM optimizes drug combinations by choosing the minimum number of drugs that produce the maximal desired intracellular effects based on nested effects modeling. We demonstrate the performance of DRUG-NEM using single-cell drug perturbation data from tumor cell lines and primary leukemia samples.

NO• /RUNX3/kynurenine metabolic signaling enhances disease aggressiveness in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy and is refractory to available treatments. Delineating the regulatory mechanisms of metabolic reprogramming, a key event in pancreatic cancer progression, may identify candidate targets with potential therapeutic significance. We hypothesized that inflammatory signaling pathways regulate metabolic adaptations in pancreatic cancer. Metabolic profiling of tumors from PDAC patients with a high- (>median, n = 31) and low-NOS2 (inducible nitric oxide synthase;

Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells.

Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.

Democratizing health system data to impact social and environmental health contexts: a novel collaborative community data-sharing model.

BACKGROUND: Community health data are infrequently viewed in the context of social and environmental health determinants. We developed a novel data-sharing model to democratize health system data and to facilitate community and population health improvement. METHODS: Durham County, the City of Durham in North Carolina, Durham health systems and other stakeholders have developed a data-sharing model to inform local community health efforts. Aggregated health system data obtained through clinical encounters are shared publicly, providing data on the prevalence of health conditions of interest to the community. RESULTS: A community-owned web platform called the Durham Neighborhood Compass provides aggregate health data (e.g. on diabetes, heart disease, stroke and other conditions of interest) in the context of neighborhood social (e.g. income distribution, education level, demographics) and environmental (e.g. housing prices, crime rates, travel routes, school quality, grocery store proximity) contexts. Health data are aggregated annually to help community stakeholders track changes in health and health contexts over time. CONCLUSIONS: The Durham Neighborhood Compass is among the first collaborative public efforts to democratize health system data in the context of social and environmental health determinants. This model could be adapted elsewhere to support local community and population health improvement initiatives.

Creation of an international registry to support discovery in schwannomatosis.

Schwannomatosis is a tumor suppressor syndrome that causes multiple tumors along peripheral nerves. Formal diagnostic criteria were first published in 2005. Variability in clinical presentation and a relative lack of awareness of the syndrome have contributed to difficulty recognizing affected individuals and accurately describing the natural history of the disorder. Many critical questions such as the mutations underlying schwannomatosis, genotype-phenotype correlations, inheritance patterns, pathologic diagnosis of schwannomatosis-associated schwannomas, tumor burden in schwannomatosis, the incidence of malignancy, and the effectiveness of current, or new treatments remain unanswered. A well-curated registry of schwannomatosis patients is needed to facilitate research in field. An international consortium of clinicians and scientists across multiple disciplines with expertise in schwannomatosis was established and charged with the task of designing and populating a schwannomatosis patient registry. The International Schwannomatosis Registry (ISR) was built around key data points that allow confirmation of the diagnosis and identification of potential research subjects to advance research to further the knowledge base for schwannomatosis. A registry with 389 participants enrolled to date has been established. Twenty-three additional subjects are pending review. A formal process has been established for scientific investigators to propose research projects, identify eligible subjects, and seek collaborators from ISR sites. Research collaborations have been created using the information collected by the registry and are currently being conducted. The ISR is a platform from which multiple research endeavors can be launched, facilitating connections between affected individuals interested in participating in research and researchers actively investigating a variety of aspects of schwannomatosis. © 2016 Wiley Periodicals, Inc.

The role of purified Clostridium difficile glucosylating toxins in disease pathogenesis utilizing a murine cecum injection model.

Most pathogenic Clostridium difficile produce two major exotoxins TcdA and TcdB, in the absence of which the bacterium is non-pathogenic. While it is important to investigate the role of each toxin in the pathogenesis of C. difficile infection (CDI) using isogenic strains, it is impossible to precisely control the expression levels of individual toxins and exclude bacterial factors that may contribute to the toxins' effects during infection. In this study, we utilized an acute intestinal disease model by injecting purified toxins directly into mouse cecum after a midline laparotomy. We evaluated the physical condition of mice by clinical score and survival, and the intestinal tissue damage and inflammation by histology. Depending on the dose of the toxins, mice developed mild to severe colitis, experienced diarrhea or rapidly died. We found that both purified TcdA and TcdB were able to induce clinical disease, intestinal inflammation, and tissue damage that resembled CDI. TcdA was significantly faster in inducing intestinal inflammation and tissue damage, and was approximately five times more potent than TcdB in terms of inducing severe gut disease and death outcomes in mice. Moreover, we found that the two toxins had significant synergistic effects on disease induction. Comparison of the in vivo toxicity of TcdB from clinical strains revealed that TcdB from an epidemic RT 027 strain was more toxic than the others. Our study thus demonstrates that both TcdA and TcdB, independent of other factors from C. difficile bacterium, are able to cause disease that resembles CDI and highlights the importance of targeting both toxins for vaccines and therapeutics against the disease.

A LUHMES 3D dopaminergic neuronal model for neurotoxicity testing allowing long-term exposure and cellular resilience analysis.

Several shortcomings of current Parkinson's disease (PD) models limit progress in identification of environmental contributions to disease pathogenesis. The conditionally immortalized cell line LUHMES promises to make human dopaminergic neuronal cultures more easily available, but these cells are difficult to culture for extended periods of time. We overcame this problem by culturing them in 3D with minor medium modifications. The 3D neuronal aggregates allowed penetration by small molecules and sufficient oxygen and nutrient supply for survival of the innermost cells. Using confocal microscopy, gene expression, and flow cytometry, we characterized the 3D model and observed a highly reproducible differentiation process. Visualization and quantification of neurites in aggregates was achieved by adding 2 % red fluorescent protein-transfected LUHMES cells. The mitochondrial toxicants and established experimental PD agents, rotenone and MPP+, perturbed genes involved in one-carbon metabolism and transsulfuration pathways (ASS1, CTH, and SHTM2) as in 2D cultures. We showed, for the first time in LUHMES, down-regulation of mir-7, a miRNA known to target alpha-synuclein and to be involved in PD. This was observed as early as 12 h after rotenone exposure, when pro-apoptotic mir-16 and rotenone-sensitive mir-210 were not yet significantly perturbed. Finally, washout experiments demonstrated that withdrawal of rotenone led to counter-regulation of mir-7 and ASS1, CTH, and SHTM2 genes. This suggests a possible role of these genes in direct cellular response to the toxicant, and the model appears to be suitable to address the processes of resilience and recovery in neurotoxicology and Parkinson's disease in future studies.

Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab.

Clostridium difficile infection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibody-mediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated with C. difficile in these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fcγ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials.

Mass cytometry to decipher the mechanism of nongenetic drug resistance in cancer.

Nongenetic resistance has recently been described as a major impediment to effective cancer therapy. Nongenetic resistance is challenging to study since it occurs nonuniformly, even in cell lines, and can involve the interplay of multiple survival pathways. Until recently, no technology allowed measurement of large-scale alterations in survival pathways with single-cell resolution. Mass cytometry, a flow-based technique in which the activation of up to 50 proteins can be measured simultaneously in single-cell, now provides the ability to examine nongenetic resistance on the functional level on a cell-by-cell basis. The application of mass cytometry, in combination with new bioinformatic techniques, will allow fundamental questions on nongenetic resistance to be addressed: Is resistance caused by selection of cells with a pre-existing survival phenotype or induction of a survival program? Which survival pathways are necessary for nongenetic resistance and how do they interact? Currently, mass cytometry is being used to investigate the mechanism of nongenetic resistance to TRAIL-induced apoptosis. The approaches being developed to understand resistance to TRAIL will likely be applied to elucidate the mechanisms of nongenetic resistance broadly and in the clinic.

Modulation of DNA-polyamide interaction by ß-alanine substitutions: a study of positional effects on binding affinity, kinetics and thermodynamics.

Hairpin polyamides (PAs) are an important class of sequence-specific DNA minor groove binders, and frequently employ a flexible motif, ß-alanine (ß), to reduce the molecular rigidity to maintain the DNA recognition register. To better understand the diverse effects that ß can have on DNA-PA binding affinity, selectivity, and especially kinetics, which have rarely been reported, we have initiated a detailed study for an eight-heterocyclic hairpin PA and its ß derivatives with their cognate and mutant sequences. With these derivatives, all internal pyrroles of the parent PA are systematically substituted with single or double ßs. A set of complementary experiments have been conducted to evaluate the molecular interactions in detail: UV-melting, biosensor-surface plasmon resonance, circular dichroism and isothermal titration calorimetry. The ß substitutions generally weaken the binding affinities of these PAs with cognate DNA, and have large and diverse influences on PA binding kinetics in a position- and number-dependent manner. The DNA base mutations have also shown positional effects on the binding of a single PA. Besides the ß substitutions, the monocationic Dp group [3-(dimethylamino)propylamine] in parent PA has been modified into a dicationic Ta group (3,3'-diamino-N-methyldipropylamine) to minimize the frequently observed PA aggregation with ITC experiments. The results clearly show that the Ta modification not only maintains the DNA binding mode and affinity of PA, but also significantly reduces PA aggregation and allows the complete thermodynamic signature of eight-ring hairpin PA to be determined for the first time. This combined set of results significantly extends our understanding of the energetic basis of specific DNA recognition by PAs.

MicroRNA-1 is a candidate tumor suppressor and prognostic marker in human prostate cancer.

We previously reported that miR-1 is among the most consistently down-regulated miRs in primary human prostate tumors. In this follow-up study, we further corroborated this finding in an independent data set and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a candidate predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair and actin dynamics. This observation was further corroborated with protein expression analysis and 3'-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that the miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters the cellular organization of F-actin and inhibits tumor cell invasion and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.

Macrophage migration inhibitory factor induces epithelial to mesenchymal transition, enhances tumor aggressiveness and predicts clinical outcome in resected pancreatic ductal adenocarcinoma.

MIF is a proinflammatory cytokine and is implicated in cancer. A higher MIF level is found in many human cancer and cancer-prone inflammatory diseases, including chronic pancreatitis and pancreatic cancer. We tested the hypothesis that MIF contributes to pancreatic cancer aggressiveness and predicts disease outcome in resected cases. Consistent with our hypothesis we found that an elevated MIF mRNA expression in tumors was significantly associated with poor outcome in resected cases. Multivariate Cox-regression analysis further showed that MIF is independently associated with patients' survival (HR = 2.26, 95% CI = 1.17-4.37, p = 0.015). Mechanistic analyses revealed that MIF overexpression decreased E-cadherin and increased vimentin mRNA and protein levels in pancreatic cancer cell lines, consistent with the features of epithelial-to-mesenchymal transition (EMT). Furthermore, MIF-overexpression significantly increased ZEB1/2 and decreased miR-200b expression, while shRNA-mediated inhibition of MIF increased E-cadherin and miR-200b expression, and reduced the expression of ZEB1/2 in Panc1 cells. Re-expression of miR-200b in MIF overexpressing cells restored the epithelial characteristics, as indicated by an increase in E-cadherin and decrease in ZEB1/2 and vimentin expression. A reduced sensitivity to the chemotherapeutic drug, gemcitabine, occurred in MIF-overexpressing cells. Indicative of an increased malignant potential, MIF over-expressing cells showed significant increase in their invasion ability in vitro, and tumor growth and metastasis in an orthotopic xenograft mouse model. These results support a role of MIF in disease aggressiveness, indicating its potential usefulness as a candidate target for designing improved treatment in pancreatic cancer.

Analysis of indoleamine 2-3 dioxygenase (IDO1) expression in breast cancer tissue by immunohistochemistry.

INTRODUCTION: The immunosuppressive enzyme, indoleamine 2,3 dioxygenase (IDO), is overexpressed in many different tumor types including breast cancer. IDO inhibitors synergize with chemotherapy in breast cancer murine models. Characterizing IDO expression in breast cancer could define which patients receive IDO inhibitors. This study analyzed IDO protein expression in 203 breast cancer cases. The relationship between IDO, overall survival (OS), disease-specific survival (DSS), clinicopathologic, molecular, and immune tumor infiltrate factors was evaluated. METHODS: Expression of IDO, estrogen receptor (ER), progesterone receptor (PR), human epithelial receptor 2, cytokeratin 5/6, epithelial growth factor receptor, phosphorylated AKT, neoangiogenesis, nitrogen oxide synthetase 2 (NOS2), cyclooxygenase 2 (COX2), FoxP3, CD8, and CD11b on archival breast cancer tissue sections was evaluated by immunohistochemistry. Associations between IDO and these markers were explored by a univariate and multivariate analysis. Survival was analyzed using Kaplan-Meier (OS) and Wilcoxon two-sample (DSS) tests. RESULTS: IDO expression was higher in ER+ tumors compared to ER- tumors. IDO was lower in those with higher neoangiogenesis. OS was better in ER+ patients with high IDO expression. DSS was better in node-positive patients with high IDO expression. IDO activity positively correlates with NOS2. COX2 as positively correlated with IDO on univariate but not multivariate analysis. There was a trend toward greater numbers of CD11b+ cells in IDO-low tumors. CONCLUSIONS: IDO protein expression is lower in ER- breast tumors with greater neoangiogenesis. Future clinical trials evaluating the synergy between IDO inhibitors and chemotherapy should take this finding into account and stratify for ER status in the trial design.

Transanal endoscopic microsurgery: a new technique for completion proctectomy.

AIM: Following subtotal colectomy, the retained rectal stump is a potential source of morbidity. Although restorative ileal pouch-anal anastomosis is the gold standard for ulcerative colitis, up to 14% of patients will opt for a permanent ileostomy and undergo completion proctectomy, traditionally by an abdomino-perineal approach, which itself carries significant morbidity. We describe a new technique of perineal proctectomy using transanal endoscopic microsurgery (TEMS) equipment. To our knowledge, this technique has not previously been described in the literature. METHOD: Twelve patients, mean (SD) age 66 (±13) years, underwent TEMS proctectomy, performed by a single surgeon between January 2007 and October 2011. Excision began with an intersphincteric dissection following which the TEMS (WOLF) proctoscope was inserted and close rectal dissection was performed, entering the peritoneal cavity (if the top of the stump was intraperitoneal). Following perineal extraction of the specimen, the external sphincter and skin were closed with an absorbable suture. RESULTS: Nine patients had inflammatory bowel disease, two had neoplasia and one had intractable radiation proctitis. The mean (SD) rectal stump length was 17.8 (±6.1) cm and the peritoneal cavity was entered in nine patients, with no small-bowel injury. The median postoperative hospital stay was 5.5 days. In four patients there was delayed healing of the perineal wound. There was no perioperative mortality. CONCLUSION: TEMS perineal proctectomy is a novel, but safe, technique that may avoid the need for a traditional abdominoperineal approach in selected patients.

Automated detection, delineation and quantification of whole-body bone metastasis using FDG-PET/CT images.

Non-small cell lung cancer (NSCLC) patients with the metastatic spread of disease to the bone have high morbidity and mortality. Stereotactic ablative body radiotherapy increases the progression free survival and overall survival of these patients with oligometastases. FDG-PET/CT, a functional imaging technique combining positron emission tomography (PET) with 18 F-fluorodeoxyglucose (FDG) and computer tomography (CT) provides improved staging and identification of treatment response. It is also associated with reduction in size of the radiotherapy tumour volume delineation compared with CT based contouring in radiotherapy, thus allowing for dose escalation to the target volume with lower doses to the surrounding organs at risk. FDG-PET/CT is increasingly being used for the clinical management of NSCLC patients undergoing radiotherapy and has shown high sensitivity and specificity for the detection of bone metastases in these patients. Here, we present a software tool for detection, delineation and quantification of bone metastases using FDG-PET/CT images. The tool extracts standardised uptake values (SUV) from FDG-PET images for auto-segmentation of bone lesions and calculates volume of each lesion and associated mean and maximum SUV. The tool also allows automatic statistical validation of the auto-segmented bone lesions against the manual contours of a radiation oncologist. A retrospective review of FDG-PET/CT scans of more than 30 candidate NSCLC patients was performed and nine patients with one or more metastatic bone lesions were selected for the present study. The SUV threshold prediction model was designed by splitting the cohort of patients into a subset of 'development' and 'validation' cohorts. The development cohort yielded an optimum SUV threshold of 3.0 for automatic detection of bone metastases using FDG-PET/CT images. The validity of the derived optimum SUV threshold on the validation cohort demonstrated that auto-segmented and manually contoured bone lesions showed strong concordance for volume of bone lesion (r = 0.993) and number of detected lesions (r = 0.996). The tool has various applications in radiotherapy, including but not limited to studies determining optimum SUV threshold for accurate and standardised delineation of bone lesions and in scientific studies utilising large patient populations for instance for investigation of the number of metastatic lesions that can be treated safety with an ablative dose of radiotherapy without exceeding the normal tissue toxicity.