Publications by authors named "Chand Khanna"

117 Publications

Leukocyte and cytokine variables in asymptomatic Pugs at genetic risk of necrotizing meningoencephalitis.

J Vet Intern Med 2021 Oct 23. Epub 2021 Oct 23.

Neurogenomics Division, Translational Genomics Research Institute, Phoenix, Arizona, USA.

Background: Necrotizing meningoencephalitis (NME, aka Pug dog encephalitis) is an inflammatory brain condition associated with advanced disease at initial presentation, rapid progression, and poor response to conventional immunomodulatory therapy.

Hypothesis/objectives: That genetic risk for NME, defined by a common germline DNA haplotype located on chromosome 12, is associated with altered blood cytokine concentrations and leukocyte subsets in asymptomatic Pugs.

Animals: Forty Pug dogs asymptomatic for NME from a hospital sample.

Methods: Prospective observational cohort study, including germline genome-wide genotyping, plasma cytokine determination by multiplexed profiling, and leukocyte subset characterization by flow cytometric analysis.

Results: Seven (18%) dogs were high risk, 10 (25%) medium risk, and 23 (58%) low risk for NME, giving a risk haplotype frequency of 30%. High and medium risk Pugs had significantly lower proportion of CD4+ T cells (median 22% [range, 7.3%-38%] vs 29% [range, 16%-41%], P = .03) and higher plasma IL-10 concentrations than low-risk Pugs (median 14.11 pg/mL [range, 9.66-344.19 pg/mL] vs 12.21 pg/mL [range, 2.59-18.53 pg/mL], P = .001). No other variables were significantly associated with the NME haplotype-based risk.

Conclusions And Clinical Importance: These data suggest an immunological underpinning to NME and a biologic rationale for future clinical trials that investigate novel diagnostic, preventative, and therapeutic strategies for this disease.
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http://dx.doi.org/10.1111/jvim.16293DOI Listing
October 2021

Assessing a Novel 3D Assay System for Drug Screening against OS Metastasis.

Pharmaceuticals (Basel) 2021 Sep 25;14(10). Epub 2021 Sep 25.

Ethos Discovery, Woburn, MA 01801, USA.

Osteosarcoma (OS) is an aggressive mesenchymal cell tumor that carries a poor long-term prognosis. Despite definitive surgery for the primary tumor and adjuvant chemotherapy, pulmonary metastasis is common and is the primary cause of morbidity. To improve outcomes for patients, we have developed and optimized a phenotypic screen for drugs that may target OS disseminated tumor cells (DTCs) and inhibit their metastatic outbreak rather than merely screening for cytotoxic activity against proliferating cells, as is commonly conducted in conventional drug discovery approaches. We report on the validation of a previously described 3D reconstituted basement membrane extract (3D BME) model system for tumor dormancy and metastatic outgrowth adapted to clonal pairs of high and low metastatic OS cells. A post-hoc validation of the assay was possible by comparing the activity of a drug in our assay with early evidence of activity in human OS clinical trials (regorafenib and saracatinib). In this validation, we found concordance between our assay and human clinical trial experience We then explored an approved veterinary small molecule inhibitor of Janus kinase-1 (oclacitinib) as a potential drug candidate to take advantage of the high prevalence of OS in pet dogs and its translational value to humans. Despite the biological rationale, we found no evidence to support the use of oclacitinib as an antimetastatic agent in OS. The findings support our 3D BME assay as a highly efficient method to examine drugs for activity in targeting OS DTCs.
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http://dx.doi.org/10.3390/ph14100971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8540451PMC
September 2021

Adjuvant Sirolimus Does Not Improve Outcome in Pet Dogs Receiving Standard-of-Care Therapy for Appendicular Osteosarcoma: A Prospective, Randomized Trial of 324 Dogs.

Clin Cancer Res 2021 Jun 22;27(11):3005-3016. Epub 2021 Mar 22.

Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts.

Purpose: The mTOR pathway has been identified as a key nutrient signaling hub that participates in metastatic progression of high-grade osteosarcoma. Inhibition of mTOR signaling is biologically achievable with sirolimus, and might slow the outgrowth of distant metastases. In this study, pet dogs with appendicular osteosarcoma were leveraged as high-value biologic models for pediatric osteosarcoma, to assess mTOR inhibition as a therapeutic strategy for attenuating metastatic disease progression.

Patients And Methods: A total of 324 pet dogs diagnosed with treatment-naïve appendicular osteosarcoma were randomized into a two-arm, multicenter, parallel superiority trial whereby dogs received amputation of the affected limb, followed by adjuvant carboplatin chemotherapy ± oral sirolimus therapy. The primary outcome measure was disease-free interval (DFI), as assessed by serial physical and radiologic detection of emergent macroscopic metastases; secondary outcomes included overall 1- and 2-year survival rates, and sirolimus pharmacokinetic variables and their correlative relationship to adverse events and clinical outcomes.

Results: There was no significant difference in the median DFI or overall survival between the two arms of this trial; the median DFI and survival for standard-of-care (SOC; defined as amputation and carboplatin therapy) dogs was 180 days [95% confidence interval (CI), 144-237] and 282 days (95% CI, 224-383) and for SOC + sirolimus dogs, it was 204 days (95% CI, 157-217) and 280 days (95% CI, 252-332), respectively.

Conclusions: In a population of pet dogs nongenomically segmented for predicted mTOR inhibition response, sequentially administered adjuvant sirolimus, although well tolerated when added to a backbone of therapy, did not extend DFI or survival in dogs with appendicular osteosarcoma.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-0315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8172450PMC
June 2021

Identification of potential modulators of osteosarcoma metastasis by high-throughput cellular screening of natural products.

Chem Biol Drug Des 2021 01 28;97(1):77-86. Epub 2020 Jul 28.

Molecular Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA.

A high-throughput screening assay was developed and applied to a large library of natural product extract samples, in order to identify compounds which preferentially inhibited the in vitro 2D growth of a highly metastatic osteosarcoma cell line (MG63.3) compared to a cognate parental cell line (MG63) with low metastatic potential. Evaluation of differentially active natural product extracts with bioassay-guided fractionation led to the identification of lovastatin (IC  = 11 µm) and the limonoid toosendanin (IC  = 26 nm). Other statins and limonoids were then tested, and cerivastatin was identified as a particularly potent (IC  < 0.1 µm) and selective agent. These compounds potently and selectively induced apoptosis in MG63.3 cells, but not MG63. Assays with other cell pairs were used to examine the generality of these results. Statins and limonoids may represent unexplored opportunities for development of modulators of osteosarcoma metastasis. As cerivastatin was previously approved for clinical use, it could be considered for repurposing in osteosarcoma, pending validation in further models.
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http://dx.doi.org/10.1111/cbdd.13762DOI Listing
January 2021

Prospective observational study of dogs with splenic mass rupture suggests potentially lower risk of malignancy and more favourable perioperative outcomes.

Vet Comp Oncol 2020 Dec 15;18(4):811-817. Epub 2020 Jun 15.

Emergency and Critical Care, Ethos Veterinary Health, Woburn, Massachusetts, USA.

Haemoperitoneum secondary to ruptured splenic tumours can be either benign or malignant in origin. The majority of previous studies of canine haemoperitoneum have been retrospective, which are associated with well-recognized biases, such as the potential to underappreciate the diversity of outcomes in a complex presentation such as haemoperitoneum. This study seeks to prospectively define perioperative morbidity and mortality of haemoperitoneum in dogs secondary to ruptured splenic masses. Forty dogs with haemoperitoneum secondary to a ruptured splenic mass met the inclusion criteria. As expected, the cohort predominately consisted of older large breed dogs. All dogs underwent preoperative staging and had a splenectomy performed. Histopathologic analysis was performed on the splenic mass, as well as any possible metastatic lesions that were noted intra-operatively. Perioperative care outside of splenectomy was delivered in specialty practices using current conventional approaches to care (eg, transfusions and anti-arrhythmic medications). Fifteen dogs (37.5%) had benign splenic tumours and were cured with surgery alone, whereas 62.5% had malignant disease (most often haemangiosarcoma [HSA]). Surgical outcomes were highly favourable in the vast majority of dogs. Indeed, 38 dogs (95%) survived and were discharged after a median hospitalization of 39.5 hours. Independent predictors of longer hospitalization times included receiving a transfusion and the development of an arrhythmia. Although small, this cohort defines distinctive and optimistic perspectives for dogs with haemoperitoneum from splenic tumour rupture. These favourable outcomes from this prospective study are sufficient to ask if larger prospective studies should be conducted to better inform owners during this challenging cancer emergency presentation.
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http://dx.doi.org/10.1111/vco.12621DOI Listing
December 2020

Provocative questions in osteosarcoma basic and translational biology: A report from the Children's Oncology Group.

Cancer 2019 10 29;125(20):3514-3525. Epub 2019 Jul 29.

Dana-Farber Cancer Institute, Boston, and Broad Institute of Harvard and MIT, Cambridge, Massachusetts.

Patients who are diagnosed with osteosarcoma (OS) today receive the same therapy that patients have received over the last 4 decades. Extensive efforts to identify more effective or less toxic regimens have proved disappointing. As we enter a postgenomic era in which we now recognize OS not as a cancer of mutations but as one defined by p53 loss, chromosomal complexity, copy number alteration, and profound heterogeneity, emerging threads of discovery leave many hopeful that an improving understanding of biology will drive discoveries that improve clinical care. Under the organization of the Bone Tumor Biology Committee of the Children's Oncology Group, a team of clinicians and scientists sought to define the state of the science and to identify questions that, if answered, have the greatest potential to drive fundamental clinical advances. Having discussed these questions in a series of meetings, each led by invited experts, we distilled these conversations into a series of seven Provocative Questions. These include questions about the molecular events that trigger oncogenesis, the genomic and epigenomic drivers of disease, the biology of lung metastasis, research models that best predict clinical outcomes, and processes for translating findings into clinical trials. Here, we briefly present each Provocative Question, review the current scientific evidence, note the immediate opportunities, and speculate on the impact that answered questions might have on the field. We do so with an intent to provide a framework around which investigators can build programs and collaborations to tackle the hardest problems and to establish research priorities for those developing policies and providing funding.
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http://dx.doi.org/10.1002/cncr.32351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948723PMC
October 2019

Towards the Delivery of Precision Veterinary Cancer Medicine.

Vet Clin North Am Small Anim Pract 2019 Sep 28;49(5):809-818. Epub 2019 Jun 28.

EthosVeterinaryHealth LLC, 20 Cabot Road, Woburn, MA 01801, USA; EthosDiscovery(501c3), Washington, DC, USA. Electronic address:

We introduce a next phase in the evolution of medicine affecting human and veterinary patients. This evolution, genomic cancer medicine (Pmed), involves expansion of genomic and molecular biology into clinical medicine. The implementation of these new technologies has already begun and is a commercial reality. We introduce the underpinnings for this evolution, and focus on application in complex disease states. Pet owners have begun requesting Pmed technologies. To meet this demand, it is important to be aware of the opportunities and obstacles associated with available Pmed offerings as well as the current state of the field.
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http://dx.doi.org/10.1016/j.cvsm.2019.04.011DOI Listing
September 2019

Polymerase chain reaction for antigen receptor rearrangement: Benchmarking performance of a lymphoid clonality assay in diverse canine sample types.

J Vet Intern Med 2019 May 2;33(3):1392-1402. Epub 2019 Apr 2.

Ethos Discovery, San Diego, California.

Background: Polymerase chain reaction for antigen receptor rearrangement (PARR) is a molecular diagnostic tool used for discrimination of lymphoid malignancies in dogs from benign processes. Assay variations have been described and are commercially available, but performance metrics are not uniformly reported.

Objectives: To describe performance (accuracy, sensitivity, specificity) and rigorous benchmarking of a PARR protocol (ePARR) in clinically relevant samples.

Animals: One hundred eighty-one client-owned dogs.

Methods: Lymphoma and benign tissues representative of the clinical spectrum with gold standard histopathologic and immunohistochemical diagnoses were collected. Assay development and benchmarking were performed on fresh frozen (FF) tissue, formalin-fixed paraffin-embedded (FFPE) tissue, flow cytometry pellets, and air-dried fine-needle aspirates (FNA). Assay performance was determined for FFPE from 56 dogs (18 B-cell lymphoma, 24 T-cell lymphoma, and 14 non-lymphoma), 80 frozen flow cytometry pellets (66 B-cell lymphoma, 14 T-cell lymphoma, 0 non-lymphoma), and 41 air-dried FNA slides (23 lymphoma, 18 non-lymphoma).

Results: For discrimination of lymphoma versus non-lymphoma, ePARR had 92% and 92% sensitivity and specificity on FFPE with 92% accuracy, 85% sensitivity from flow cytometry pellets (non-lymphoma was not evaluated to calculate specificity) with 85% accuracy, and 100% and 100% sensitivity and specificity for FNA with 100% accuracy. Stringent quality control criteria decreased assay success rate without significant performance improvement. Performance metrics were lower in most cases for discrimination of B- or T-cell versus non-B- or non-T-cell samples than for lymphoma versus non-lymphoma.

Conclusions And Clinical Importance: These benchmarking data facilitate effective interpretation and application of PARR assays in multiple sample types.
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http://dx.doi.org/10.1111/jvim.15485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524097PMC
May 2019

Somatic inactivating PTPRJ mutations and dysregulated pathways identified in canine malignant melanoma by integrated comparative genomic analysis.

PLoS Genet 2018 09 6;14(9):e1007589. Epub 2018 Sep 6.

Integrated Cancer Genomics Division, Translational Genomics Research Institute (TGen), Phoenix, Arizona, United States of America.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.
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http://dx.doi.org/10.1371/journal.pgen.1007589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126841PMC
September 2018

NCI Comparative Oncology Program Testing of Non-Camptothecin Indenoisoquinoline Topoisomerase I Inhibitors in Naturally Occurring Canine Lymphoma.

Clin Cancer Res 2018 12 30;24(23):5830-5840. Epub 2018 Jul 30.

Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.

Purpose: Only one chemical class of topoisomerase I (TOP1) inhibitors is FDA approved, the camptothecins with irinotecan and topotecan widely used. Because of their limitations (chemical instability, drug efflux-mediated resistance, and diarrhea), novel TOP1 inhibitors are warranted. Indenoisoquinoline non-camptothecin topoisomerase I (TOP1) inhibitors overcome chemical instability and drug resistance that limit camptothecin use. Three indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan), and LMP744, were examined in a phase I study for lymphoma-bearing dogs to evaluate differential efficacy, pharmacodynamics, toxicology, and pharmacokinetics.

Experimental Design: Eighty-four client-owned dogs with lymphomas were enrolled in dose-escalation cohorts for each indenoisoquinoline, with an expansion phase for LMP744. Efficacy, tolerability, pharmacokinetics, and target engagement were determined.

Results: The MTDs were 17.5 mg/m for LMP 776 and 100 mg/m for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m LMP400 was well-tolerated and MTD was not reached. None of the drugs induced notable diarrhea. Sustained tumor accumulation was observed for LMP744; γH2AX induction was demonstrated in tumors 2 and 6 hours after treatment; a decrease in TOP1 protein was observed in most lymphoma samples across all compounds and dose levels, which is consistent with the fact that tumor response was also observed at low doses LMP744. Objective responses were documented for all indenoisoquinolines; efficacy (13/19 dogs) was greatest for LMP744.

Conclusions: These results demonstrate proof-of-mechanism for indenoisoquinoline TOP1 inhibitors supporting their further clinical development. They also highlight the value of the NCI Comparative Oncology Program (https://ccr.cancer.gov/Comparative-Oncology-Program) for evaluating novel therapies in immunocompetent pets with cancers.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312717PMC
December 2018

Tolerability of oral sorafenib in pet dogs with a diagnosis of cancer.

Vet Med (Auckl) 2017 8;8:97-102. Epub 2017 Dec 8.

The Oncology Service, LLC, Leesburg, VA, USA,

Sorafenib is a multi-target small molecule inhibitor of the RAF kinase family and VEGFR-2/PDGFR. The US Food and Drug Administration approved sorafenib in human patients with liver, thyroid, or renal carcinoma. The aim of this study was to help guide future pharmacokinetic (PK) studies of sorafenib in dogs with a cancer diagnosis. Client-owned dogs were eligible if they had a cytologic or histologic diagnosis of cancer. Patients were enrolled at escalating doses of sorafenib. Patients were evaluable for the study if they received at least one dose of sorafenib and presented 1 week later for a follow-up examination, blood work, and assessment of drug tolerability. The goal of this study was not to define a maximum tolerated dose as may be reasonable in conventional cytotoxic chemotherapy drugs, but rather to describe the tolerability of this drug in dogs with a cancer diagnosis, as a prequel to future sorafenib PK studies. No patients in the study had any evidence of adverse events that were attributable to sorafenib. Doses of 3 mg/kg were well tolerated and associated with a suggestion of clinical activity, supportive of future PK, and pharmacodynamic analysis. Such future studies are recommended at this dose to define the associated exposure achieved and determine a reasonable schedule for sorafenib administration.
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http://dx.doi.org/10.2147/VMRR.S149678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042486PMC
December 2017

Leveraging Comparative Oncology in the Hopes of Improving Therapies for Breast Cancer.

Authors:
Chand Khanna

Semin Oncol 2017 08 23;44(4):301. Epub 2017 Nov 23.

Ethos Veterinary Health, Woburn, MA; Ethos Discovery, Washington, DC.

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http://dx.doi.org/10.1053/j.seminoncol.2017.11.003DOI Listing
August 2017

Positively selected enhancer elements endow osteosarcoma cells with metastatic competence.

Nat Med 2018 02 15;24(2):176-185. Epub 2018 Jan 15.

Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio, USA.

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.
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http://dx.doi.org/10.1038/nm.4475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803371PMC
February 2018

Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.

PLoS One 2017 19;12(9):e0185089. Epub 2017 Sep 19.

Tumor Metastasis Biology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

Activation of the unfolded protein response (UPR) in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus) and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185089PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605117PMC
October 2017

Osteosarcoma enters a post genomic era with in silico opportunities: Generation of the High Dimensional Database for facilitating sarcoma biology research: A report from the Children's Oncology Group and the QuadW Foundation.

PLoS One 2017 21;12(7):e0181204. Epub 2017 Jul 21.

Department of Veterinary Clinical Medicine, University of Illinois, Urbana, Illinois, United States of America.

The prospective banking of osteosarcoma tissue samples to promote research endeavors has been realized through the establishment of a nationally centralized biospecimen repository, the Children's Oncology Group (COG) biospecimen bank located at the Biopathology Center (BPC)/Nationwide Children's Hospital in Columbus, Ohio. Although the physical inventory of osteosarcoma biospecimens is substantive (>15,000 sample specimens), the nature of these resources remains exhaustible. Despite judicious allocation of these high-value biospecimens for conducting sarcoma-related research, a deeper understanding of osteosarcoma biology, in particular metastases, remains unrealized. In addition the identification and development of novel diagnostics and effective therapeutics remain elusive. The QuadW-COG Childhood Sarcoma Biostatistics and Annotation Office (CSBAO) has developed the High Dimensional Data (HDD) platform to complement the existing physical inventory and to promote in silico hypothesis testing in sarcoma biology. The HDD is a relational biologic database derived from matched osteosarcoma biospecimens in which diverse experimental readouts have been generated and digitally deposited. As proof-of-concept, we demonstrate that the HDD platform can be utilized to address previously unrealized biologic questions though the systematic juxtaposition of diverse datasets derived from shared biospecimens. The continued population of the HDD platform with high-value, high-throughput and mineable datasets allows a shared and reusable resource for researchers, both experimentalists and bioinformatics investigators, to propose and answer questions in silico that advance our understanding of osteosarcoma biology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181204PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5521774PMC
October 2017

Metabolomics uncovers a link between inositol metabolism and osteosarcoma metastasis.

Oncotarget 2017 Jun;8(24):38541-38553

Comparative Oncology Program, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.

Cancer development and progression are characterized by complex molecular events. The acquisition of these events is primarily believed to result from alterations in gene and protein expression/function. Recent studies have also suggested the role of metabolic alterations, or "metabolic reprogramming," that may similarly contribute to these events. Indeed, our previous investigations in osteosarcoma (OS) identified metabolic changes uniquely linked to metastasis. Based on those findings, here we sought to build a more detailed understanding of the specific alterations in metabolites or metabolic pathways that may be responsible for the observed metastasis-associated metabolic alterations, suggested by gene expression data. This was pursued using a combination of high-throughput liquid- and gas-chromatography-based mass spectrometry (LC/MS and GC/MS) for a global metabolic profiling/subtraction of four pairs of high/low metastatic OS cell lines. By comparing the identity and level of the metabolites between high/low metastatic cells, several metabolic pathways were identified to be differentially activated, such as arginine, glutathione, inositol and fatty acid metabolic pathways. To further interrogate these results, we investigated the effects of inositol pathway dysregulation, through the exposure of metastatic OS cells to IP6 (inositol hexaphosphate). Although IP6 exposures had modest to minimal effects on cell proliferation, we observed reduced cellular glycolysis, down-regulation of PI3K/Akt signaling and suppression of OS metastatic progression. Collectively these data supported further investigation of metabolic sensitivities as anti-metastatic strategies in a clinical setting as well as investigation of altered metabolomics associated with metastatic progression.
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http://dx.doi.org/10.18632/oncotarget.15872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503552PMC
June 2017

Upregulation of Glucose-Regulated Protein 78 in Metastatic Cancer Cells Is Necessary for Lung Metastasis Progression.

Neoplasia 2016 11 28;18(11):699-710. Epub 2016 Oct 28.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; Ethos Discovery in Washington DC and Ethos Veterinary Health, Wolburn MA, USA. Electronic address:

Metastasis is the cause of more than 90% of all cancer deaths. Despite this fact, most anticancer therapeutics currently in clinical use have limited efficacy in treating established metastases. Here, we identify the endoplasmic reticulum chaperone protein, glucose-regulated protein 78 (GRP78), as a metastatic dependency in several highly metastatic cancer cell models. We find that GRP78 is consistently upregulated when highly metastatic cancer cells colonize the lung microenvironment and that mitigation of GRP78 upregulation via short hairpin RNA or treatment with the small molecule IT-139, which is currently under clinical investigation for the treatment of primary tumors, inhibits metastatic growth in the lung microenvironment. Inhibition of GRP78 upregulation and an associated reduction in metastatic potential have been shown in four highly metastatic cell line models: three human osteosarcomas and one murine mammary adenocarcinoma. Lastly, we show that downmodulation of GRP78 in highly metastatic cancer cells significantly increases median survival times in our in vivo animal model of experimental metastasis. Collectively, our data indicate that GRP78 is an attractive target for the development of antimetastatic therapies.
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http://dx.doi.org/10.1016/j.neo.2016.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5094383PMC
November 2016

mTOR Inhibition Mitigates Enhanced mRNA Translation Associated with the Metastatic Phenotype of Osteosarcoma Cells In Vivo.

Clin Cancer Res 2016 Dec 24;22(24):6129-6141. Epub 2016 Jun 24.

Pediatric Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland.

Purpose: To successfully metastasize, tumor cells must respond appropriately to biological stressors encountered during metastatic progression. We sought to test the hypothesis that enhanced efficiency of mRNA translation during periods of metastatic stress is required for metastatic competence of osteosarcoma and that this metastasis-specific adaptation is amenable to therapeutic intervention.

Experimental Design: We employ novel reporter and proteomic systems that enable tracking of mRNA translation efficiency and output in metastatic osteosarcoma cells as they colonize the lungs. We test the potential to target mRNA translation as an antimetastatic therapeutic strategy through pharmacokinetic studies and preclinical assessment of the prototypic mTOR inhibitor, rapamycin, across multiple models of metastasis.

Results: Metastatic osteosarcoma cells translate mRNA more efficiently than nonmetastatic cells during critical stressful periods of metastatic colonization of the lung. Rapamycin inhibits translational output during periods of metastatic stress, mitigates lung colonization, and prolongs survival. mTOR-inhibiting exposures of rapamycin are achievable in mice using treatment schedules that correspond to human doses well below the MTDs defined in human patients, and as such are very likely to be tolerated over long exposures alone and in combination with other agents.

Conclusions: Metastatic competence of osteosarcoma cells is dependent on efficient mRNA translation during stressful periods of metastatic progression, and the mTOR inhibitor, rapamycin, can mitigate this translation and inhibit metastasis in vivo Our data suggest that mTOR pathway inhibitors should be reconsidered in the clinic using rationally designed dosing schedules and clinical metrics related to metastatic progression. Clin Cancer Res; 22(24); 6129-41. ©2016 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-16-0326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161706PMC
December 2016

Current state of pediatric sarcoma biology and opportunities for future discovery: A report from the sarcoma translational research workshop.

Cancer Genet 2016 05 5;209(5):182-94. Epub 2016 Apr 5.

Moffitt Cancer Center, Sarcoma Department, Adolescent and Young Adult Program, Tampa, FL, USA.

Sarcomas are a rare subgroup of pediatric cancers comprised of a variety of bone and soft-tissue tumors. While significant advances have been made in improving outcomes of patients with localized pediatric sarcomas since the addition of systemic chemotherapy to local control many decades ago, outcomes for patients with metastatic and relapsed sarcoma remain poor with few novel therapeutics identified to date. With the advent of new technologies to study cancer genomes, transcriptomes and epigenomes, our understanding of sarcoma biology has improved tremendously in a relatively short period of time. However, much remains to be accomplished in this arena especially with regard to translating all of this new knowledge to the bedside. To this end, a meeting was convened in Philadelphia, PA, on April 18, 2015 sponsored by the QuadW foundation, Children's Oncology Group and CureSearch for Children's Cancer that brought together sarcoma clinicians and scientists from North America to review the current state of pediatric sarcoma biology and ongoing/planned genomics based clinical trials in an effort to identify and bridge knowledge gaps that continue to exist at present. At the conclusion of the workshop, three key objectives that would significantly further our understanding of sarcoma were identified and a proposal was put forward to develop an all-encompassing pediatric sarcoma biology protocol that would address these specific needs. This review summarizes the proceedings of the workshop.
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http://dx.doi.org/10.1016/j.cancergen.2016.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497490PMC
May 2016

A RUNX2-Mediated Epigenetic Regulation of the Survival of p53 Defective Cancer Cells.

PLoS Genet 2016 Feb 29;12(2):e1005884. Epub 2016 Feb 29.

Cancer and Stem Cell Epigenetics Section, Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

The inactivation of p53 creates a major challenge for inducing apoptosis in cancer cells. An attractive strategy is to identify and subsequently target the survival signals in p53 defective cancer cells. Here we uncover a RUNX2-mediated survival signal in p53 defective cancer cells. The inhibition of this signal induces apoptosis in cancer cells but not non-transformed cells. Using the CRISPR technology, we demonstrate that p53 loss enhances the apoptosis caused by RUNX2 knockdown. Mechanistically, RUNX2 provides the survival signal partially through inducing MYC transcription. Cancer cells have high levels of activating histone marks on the MYC locus and concomitant high MYC expression. RUNX2 knockdown decreases the levels of these histone modifications and the recruitment of the Menin/MLL1 (mixed lineage leukemia 1) complex to the MYC locus. Two inhibitors of the Menin/MLL1 complex induce apoptosis in p53 defective cancer cells. Together, we identify a RUNX2-mediated epigenetic mechanism of the survival of p53 defective cancer cells and provide a proof-of-principle that the inhibition of this epigenetic axis is a promising strategy to kill p53 defective cancer cells.
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http://dx.doi.org/10.1371/journal.pgen.1005884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4771715PMC
February 2016

A Comparative Oncology Study of Iniparib Defines Its Pharmacokinetic Profile and Biological Activity in a Naturally-Occurring Canine Cancer Model.

PLoS One 2016 11;11(2):e0149194. Epub 2016 Feb 11.

Comparative Oncology Program, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

Development of iniparib as an anti-cancer agent was hindered in part by lingering questions regarding its mechanism of action, the activity of its metabolites, and their potential accumulation in tumors. Due to strong similarities in metabolism of iniparib between humans and dogs, a veterinary clinical trial in pet dogs with spontaneous cancers was designed to answer specific questions pertaining to pharmacokinetic exposures and tolerability of iniparib. Dogs were treated with iniparib alone and in combination with carboplatin chemotherapy. Iniparib doses ranged between 10-70 mg/kg intravenously (IV). Plasma, tumor and normal tissue samples were collected before and at various time points scheduled after exposure for pharmacokinetic and biologic analysis. The primary endpoints included characterization of dose-limiting toxicities (DLT) and determination of the drug exposures that could be achieved in both normal and tumor tissues. Nineteen dogs were treated. DLT included fever, anorexia, diarrhea, neutropenia, and thrombocytopenia; most effects were attributable to carboplatin based on the timing of adverse event onset. The maximum tolerated dose (MTD) of iniparib was not identified. Moderate to high variability in plasma exposure was noted for iniparib and all metabolites between animals. When quantifiable, iniparib and metabolite plasma:tumor ratios were < 0.088 and <1.7, respectively. In this study, iniparib was well tolerated as a single agent and in combination with carboplatin over a range of doses. However, clinically relevant concentrations of the parent drug and selected metabolites were not detectable in canine tumor tissues at any studied dose, thus eliminating expectations for clinical responses in dogs or humans. Negative clinical trials in humans, and the uncertainties of its mechanism of action, ultimately led to the decision to stop clinical development of the drug. Nevertheless, the questions that can be asked and answered within the comparative oncology approach are evident from this successfully executed comparative clinical trial and exemplify the value of such studies in drug development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149194PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751284PMC
July 2016

Defining the Value of a Comparative Approach to Cancer Drug Development.

Clin Cancer Res 2016 05 28;22(9):2133-8. Epub 2015 Dec 28.

Comparative Oncology Program, Center for Cancer Research, NCI, NIH, Bethesda, Maryland.

Comparative oncology as a tool in drug development requires a deeper examination of the value of the approach and examples of where this approach can satisfy unmet needs. This review seeks to demonstrate types of drug development questions that are best answered by the comparative oncology approach. We believe common perceived risks of the comparative approach relate to uncertainty of how regulatory bodies will prioritize or react to data generated from these unique studies conducted in diseased animals, and how these new data will affect ongoing human clinical trials. We contend that it is reasonable to consider these data as potentially informative and valuable to cancer drug development, but as supplementary to conventional preclinical studies and human clinical trials particularly as they relate to the identification of drug-associated adverse events. Clin Cancer Res; 22(9); 2133-8. ©2015 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111620PMC
May 2016

Pharmacokinetics of orally administered low-dose rapamycin in healthy dogs.

Am J Vet Res 2016 Jan;77(1):65-71

Objective: To determine the pharmacokinetics of orally administered rapamycin in healthy dogs.

Animals: 5 healthy purpose-bred hounds.

Procedures: The study consisted of 2 experiments. In experiment 1, each dog received rapamycin (0.1 mg/kg, PO) once; blood samples were obtained immediately before and at 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 hours after administration. In experiment 2, each dog received rapamycin (0.1 mg/kg, PO) once daily for 5 days; blood samples were obtained immediately before and at 3, 6, 24, 27, 30, 48, 51, 54, 72, 75, 78, 96, 96.5, 97, 98, 100, 102, 108, 120, 144, and 168 hours after the first dose. Blood rapamycin concentration was determined by a validated liquid chromatography-tandem mass spectrometry assay. Pharmacokinetic parameters were determined by compartmental and noncompartmental analyses.

Results: Mean ± SD blood rapamycin terminal half-life, area under the concentration-time curve from 0 to 48 hours after dosing, and maximum concentration were 38.7 ± 12.7 h, 140 ± 23.9 ng•h/mL, and 8.39 ± 1.73 ng/mL, respectively, for experiment 1, and 99.5 ± 89.5 h, 126 ± 27.1 ng•h/mL, and 5.49 ± 1.99 ng/mL, respectively, for experiment 2. Pharmacokinetic parameters for rapamycin after administration of 5 daily doses differed significantly from those after administration of 1 dose.

Conclusions And Clinical Relevance: Results indicated that oral administration of low-dose (0.1 mg/kg) rapamycin to healthy dogs achieved blood concentrations measured in nanograms per milliliter. The optimal dose and administration frequency of rapamcyin required to achieve therapeutic effects in tumor-bearing dogs, as well as toxicity after chronic dosing, need to be determined.
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http://dx.doi.org/10.2460/ajvr.77.1.65DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5642271PMC
January 2016

Letter to the Editor.

J Vet Intern Med 2016 Jan-Feb;30(1). Epub 2015 Nov 30.

Veterinary Cancer Group of Orange County, Tustin, CA.

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http://dx.doi.org/10.1111/jvim.13790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4913650PMC
May 2016

CCL9 Induced by TGFβ Signaling in Myeloid Cells Enhances Tumor Cell Survival in the Premetastatic Organ.

Cancer Res 2015 Dec 19;75(24):5283-98. Epub 2015 Oct 19.

Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland.

Tumor cell survival in the hostile distant organ is a rate-limiting step in cancer metastasis. Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, it is unclear whether these myeloid cells in the premetastatic site have any direct effect on tumor cell survival. Here, we report that chemokine CCL9 was highly induced in Gr-1(+)CD11b(+) immature myeloid cells and in premetastatic lung in tumor-bearing mice. Knockdown of CCL9 in myeloid cells decreased tumor cell survival and metastasis. Importantly, CCL9 overexpression in myeloid cells lacking TGFβ signaling rescued the tumor metastasis defect observed in mice with myeloid-specific Tgfbr2 deletion. The expression level of CCL23, the human orthologue for CCL9, in peripheral blood mononuclear cells correlated with progression and survival of cancer patients. Our study demonstrates that CCL9 could serve as a good candidate for anti-metastasis treatment by targeting the rate-limiting step of cancer cell survival. In addition, targeting CCL9 may avoid the adverse effects of TGFβ-targeted therapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-2282-TDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120555PMC
December 2015

Comparative Gene Expression Analyses Identify Luminal and Basal Subtypes of Canine Invasive Urothelial Carcinoma That Mimic Patterns in Human Invasive Bladder Cancer.

PLoS One 2015 9;10(9):e0136688. Epub 2015 Sep 9.

Department of Veterinary Clinical Sciences, Purdue University College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, United States of America; Purdue Oncological Sciences Center, Purdue University, West Lafayette, Indiana, United States of America; Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana, United States of America.

More than 160,000 people are expected to die from invasive urothelial carcinoma (iUC) this year worldwide. Research in relevant animal models is essential to improving iUC management. Naturally-occurring canine iUC closely resembles human iUC in histopathology, metastatic behavior, and treatment response, and could provide a relevant model for human iUC. The molecular characterization of canine iUC, however, has been limited. Work was conducted to compare gene expression array results between tissue samples from iUC and normal bladder in dogs, with comparison to similar expression array data from human iUC and normal bladder in the literature. Considerable similarities between enrichment patterns of genes in canine and human iUC were observed. These included patterns mirroring basal and luminal subtypes initially observed in human breast cancer and more recently noted in human iUC. Canine iUC samples also exhibited enrichment for genes involved in P53 pathways, as has been reported in human iUC. This is particularly relevant as drugs targeting these genes/pathways in other cancers could be repurposed to treat iUC, with dogs providing a model to optimize therapy. As part of the validation of the results and proof of principal for evaluating individualized targeted therapy, the overexpression of EGFR in canine bladder iUC was confirmed. The similarities in gene expression patterns between dogs and humans add considerably to the value of naturally-occurring canine iUC as a relevant and much needed animal model for human iUC. Furthermore, the finding of expression patterns that cross different pathologically-defined cancers could allow studies of dogs with iUC to help optimize cancer management across multiple cancer types. The work is also expected to lead to a better understanding of the biological importance of the gene expression patterns, and the potential application of the cross-species comparisons approach to other cancer types as well.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136688PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564191PMC
May 2016

Osteosarcoma Genetics and Epigenetics: Emerging Biology and Candidate Therapies.

Crit Rev Oncog 2015 ;20(3-4):173-97

Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

Osteosarcoma is the most common primary malignancy of bone, typically presenting in the first or second decade of life. Unfortunately, clinical outcomes for osteosarcoma patients have not substantially improved in over 30 years. This stagnation in therapeutic advances is perhaps explained by the genetic, epigenetic, and biological complexities of this rare tumor. In this review we provide a general background on the biology of osteosarcoma and the clinical status quo. We go on to enumerate the genetic and epigenetic defects identified in osteosarcoma. Finally, we discuss ongoing large-scale studies in the field and potential new therapies that are currently under investigation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894524PMC
http://dx.doi.org/10.1615/critrevoncog.2015013713DOI Listing
June 2016
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