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Purpose: A persistent issue in cancer drug development is the discordance between robust antitumor drug activity observed in laboratory models and the limited benefit frequently observed when patients are treated with the same agents in clinical trials. Difficulties in accurately modeling the complexities of human tumors may underlie this problem. To address this issue, we developed Comparative In Vivo Oncology (CIVO), which enables in situ investigation of multiple microdosed drugs simultaneously in a patient's tumor. This study was designed to test CIVO's safety and feasibility in patients with soft tissue sarcoma (STS). Patients and Methods: We conducted a single arm, prospective, 13-patient pilot study. Patients scheduled for incisional biopsy or tumor resection were CIVO-injected 1 to 3 days prior to surgery. Saline or microdoses of anticancer agents were percutaneously injected into the tumor in a columnar fashion through each of eight needles. Following excision, drug responses were evaluated in the injected tissue. Results: The primary objective was met, establishing CIVO's feasibility and safety. Device-related adverse events were limited to transient grade 1 nonserious events. In addition, biomarker evaluation of localized tumor response to CIVO microinjected drugs by IHC or with NanoString GeoMx Digital Spatial Profiler demonstrated consistency with known mechanisms of action of each drug, impact on the tumor microenvironment, and historic clinical activity. Conclusions: These results are an advance toward use of CIVO as a translational research tool for early evaluation of investigational agents and drug combinations in a novel approach to phase 0 trials. See related commentary by Sleijfer and Lolkema, p. 3897

Spontaneously occurring soft tissue sarcoma (STS) is relatively common in canine cancer patients. Because of the similarities to human disease, canine STSs are a valuable and readily available resource for the study of new therapeutics. In this study, a canine patient-derived xenograft (PDX) model, CDX-STS2, was established. The CDX-STS2 model was engrafted and expanded for systemic administration studies with chemotherapeutic agents commonly used to treat STS, including doxorubicin, docetaxel and gemcitabine. Immunohistochemistry for drug-specific biomarkers and tumour growth measurement revealed tumour sensitivity to doxorubicin and docetaxel, whereas gemcitabine had no effect on tumour growth. Although many human PDX tumour models have been established, relatively few canine PDX models have been reported to date. CDX-STS2 represents a new STS PDX research model of canine origin that will be useful in bridging preclinical research with clinical studies of STS in pet dogs.

Background: CIVO microdosing studies performed in patient tumors in situ allow drug developers to assess localized tumor and microenvironment responses to multiple agents without having to expose patients to high systemic drug levels. By concentrating microdoses of multiple different drugs within a living tumor in situ, it is possible to compare tumor and immune responses in spatially resolved regions of the same tumor. This type of early phase (Phase 0) clinical study represents a new path for drug developers to gain insight into drug efficacy, tumor associated immune cell modulation, biomarker discovery and validation, and microenvironment interactions for new drugs earlier in the drug development process. Traditionally, analysis of FFPE samples from these microdosing studies involve routine immunohistochemistry, immunofluorescence, and in situ assays that reveal changes in protein and RNA expression. These assays offer tissue wide protein and gene expression information but have limited multiplexing capabilities and dynamic range, as well as rapidly consume precious trial samples. Novel technologies such as NanoString’s GeoMxTM Digital Spatial Profiler (DSP) enable high-plex spatially resolved analysis of proteins and RNA transcripts in single FFPE tissue sections. In this proof of principle study, we utilized GeoMxTM Digital Spatial Profiler for protein and RNA expression on single FFPE sections from patient sarcoma tumors that were microdosed with multiple FDA approved drugs. Methods: Single FFPE sections from microdosed patient tumor samples were IF stained for DNA and CD3 and whole slide imaged. 600-micron and 100-micron diameter regions of interests (ROIs) were selected for DSP analysis within drug and control microinjection sites. Imaging and barcode counts were performed using GeoMxTM DSP and nCounter systems. Results: DSP protein analysis highlighted phospho-S6 and phospho-ERK upregulation in response to doxorubicin compared to vehicle site. Additionally, ROIs sampled along the doxorubicin exposure gradient showed a dose-dependent reduction of phosphorylation of both S6 and ERK proteins. DSP RNA analysis revealed drug specific transcript regulation of multiple genes in microdosed tumors, including upregulation of chemokines CXCL9 and CXCL10 at sites of doxorubicin and aldesleukin injection but not to other chemotherapy agents. Conclusions: Early phase CIVO microdosing studies combined with high-plex DSP opens the door to generating multi-omics data for multiple microdosed drugs within small patient studies. Analysis of protein and RNA expression using DSP enabled collection of targeted region of interest high density data from single FFPE sections, conserving precious patient biopsy samples. Through continued expansion of the GeoMxTM DSP analyte panels, collecting an ever-increasing depth of protein and gene expression data is possible in Phase 0 CIVO microdosing studies. Citation Format: Gary B. Deutsch, Seth M. Pollack, Matthew J. Thompson, Kenneth R. Gundle, Jessica A. Bertout, Jason P. Frazier, Emily Beirne, Marc O. Grenley, JingJing Gong, Yan Liang, Joseph M. Beechem, Richard A. Klinghoffer, Robert G. Maki. High-plex spatial profiling analysis of multidrug CIVO microdose studies in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2155.

Acute pulmonary embolism (PE) in the setting of a Patent Foramen Ovale (PFO) and clot-in-transit across the PFO poses a therapeutic dilemma. 64 year old male presented with acute dyspnea 3 weeks after ischemic stroke. CT PE-protocol revealed dilated right ventricle (RV) and proximal bilateral

Acute Pulmonary Embolism in the setting of a patent foramen ovale (PFO) poses a risk for paradoxical embolism and complicates therapeutic approach. Real time, multidisciplinary assessment facilitates appropriate care. An 18 year old female presented with syncope 1 month after anterior cruciate

The vision of a precision medicine–guided approach to novel cancer drug development is challenged by high intratumor heterogeneity and interpatient diversity. This complexity is rarely modeled accurately during preclinical drug development, hampering predictions of clinical drug efficacy. To address this issue, we developed Comparative In Vivo Oncology (CIVO) arrayed microinjection technology to test tumor responsiveness to simultaneous microdoses of multiple drugs directly in a patient's tumor. Here, in a study of 18 canine patients with soft tissue sarcoma (STS), CIVO captured complex, patient-specific tumor responses encompassing both cancer cells and multiple immune infiltrates following localized exposure to different chemotherapy agents. CIVO also classified patient-specific tumor resistance to the most effective agent, doxorubicin, and further enabled assessment of a preclinical autophagy inhibitor, PS-1001, to reverse doxorubicin resistance. In a CIVO-identified subset of doxorubicin-resistant tumors, PS-1001 resulted in enhanced antitumor activity, increased infiltration of macrophages, and skewed this infiltrate toward M1 polarization. The ability to evaluate and cross-compare multiple drugs and drug combinations simultaneously in living tumors and across a diverse immunocompetent patient population may provide a foundation from which to make informed drug development decisions. This method also represents a viable functional approach to complement current precision oncology strategies. Cancer Res; 77(11); 2869–80. ©2017 AACR.

While advances in high-throughput screening have resulted in increased ability to identify synergistic anti-cancer drug combinations, validation of drug synergy in the in vivo setting and prioritization of combinations for clinical development remain low-throughput and resource intensive. Furthermore, there is currently no viable method for prospectively assessing drug synergy directly in human patients in order to potentially tailor therapies. To address these issues we have employed the previously described CIVO platform and developed a quantitative approach for investigating multiple combination hypotheses simultaneously in single living tumors. This platform provides a rapid, quantitative and cost effective approach to compare and prioritize drug combinations based on evidence of synergistic tumor cell killing in the live tumor context. Using a gemcitabine resistant model of pancreatic cancer, we efficiently investigated nine rationally selected Abraxane-based combinations employing only 19 xenografted mice. Among the drugs tested, the BCL2/BCLxL inhibitor ABT-263 was identified as the one agent that synergized with Abraxane® to enhance acute induction of localized apoptosis in this model of human pancreatic cancer. Importantly, results obtained with CIVO accurately predicted the outcome of systemic dosing studies in the same model where superior tumor regression induced by the Abraxane/ABT-263 combination was observed compared to that induced by either single agent. This supports expanded use of CIVO as an in vivo platform for expedited in vivo drug combination validation and sets the stage for performing toxicity-sparing drug combination studies directly in cancer patients with solid malignancies.

A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.

Purpose: Mammalian target of rapamycin (mTOR) inhibition activates compensatory insulin–like growth factor receptor (IGFR) signaling. We evaluated the ridaforolimus (mTOR inhibitor) and dalotuzumab (anti-IGF1R antibody) combination. Experimental Design: In vitro and in vivo models, and a phase I study in which patients with advanced cancer received ridaforolimus (10–40 mg/day every day × 5/week) and dalotuzumab (10 mg/kg/week or 7.5 mg/kg/every other week) were explored. Results: Preclinical studies demonstrated enhanced pathway inhibition with ridaforolimus and dalotuzumab. With 87 patients treated in the phase I study, main dose-limiting toxicities (DLT) of the combination were primarily mTOR-related stomatitis and asthenia at doses of ridaforolimus lower than expected, suggesting blockade of compensatory pathways in normal tissues. Six confirmed partial responses were reported (3 patients with breast cancer); 10 of 23 patients with breast cancer and 6 of 11 patients with ER+/high-proliferative breast cancer showed antitumor activity. Conclusions: Our study provides proof-of-concept that inhibiting the IGF1R compensatory response to mTOR inhibition is feasible with promising clinical activity in heavily pretreated advanced cancer, particularly in ER+/high-proliferative breast cancer (ClinicalTrials.gov identifier: NCT00730379). Clin Cancer Res; 21(1); 49–59. ©2014 AACR.

We report the initial clinical findings of a device platform to test multiple therapeutic options simultaneously in individual living tumors within a clinical patient population, detailing for the first time comparative effects of different drugs within individual intact tumors. Most early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent the disease they are intended to represent and have a poor track record for predicting success in subsequent clinical trials. To enable in vivo analysis of anti-cancer agent efficacy at earlier stages of drug development, and to potentially enable toxicity-sparing assessment of novel agents in the oncology clinic, we have developed a technology platform called CIVO™. CIVO allows for simultaneous assessment of up to eight drugs or drug combinations in a single solid tumor while the tumor is still in the patient. Controlled microinjection-based delivery of doxorubicin, docetaxel, mafosfamide, and gemcitabine was tested in twelve patients in the canine sarcoma clinic. Drugs were co-injected in a columnar array with UV fluorescent beads resulting in easy-to-identify bands of drug at 95% of the injection sites, each at a distinct position of the patient's tumor. Tumors were resected 72h following microinjection and were subjected to multiplexed analyses for tumor response which included drug-induced ablation of sarcoma cells, apoptosis, DNA damage, mitotic arrest, immune infiltration, and feedback activation of oncogenic pathways. The CIVO-introduced drug microdoses induced spatially-defined graded, and mechanism-specific cellular changes around sites of drug exposure in a drug and patient-specific manner. Consistent with the use of doxorubicin as first line therapy in the soft tissue sarcoma clinic, the frequency and extent of response of localized tumor kill induced by Doxorubicin (6/9 patients) exceeded those of all other agents tested, with Docetaxel being next most effective (2/6 patients), and Gemcitabine being the least effective (0/9 patients). Interestingly, almost all confirmed sites of response showed evidence of mTOR pathway upregulation, suggesting that mTOR pathway activation represents a potential common mechanism of chemo-resistance across sarcoma patients exists to multiple chemo-toxic agents. We are currently exploring combinations of 1st line sarcoma drugs with mTOR inhibitors in the canine sarcoma clinic with CIVO. This data represents the first time differential effects of multiple drugs have been captured within individual intact tumors in a clinical population. Along with early responses observed in the human clinic, this sets the stage for application of this technology to identify which novel agents are likely to succeed or fail in subsequent clinical trials. Citation Format: Alicia Moreno Gonzalez, Jason Frazier, William Kerwin, Jessica Bertout, Joseph Casalini, Sally Ditzler, Nathan Caffo, Richard A. Klinghoffer. A platform to test multiple therapies simultaneously in the intact tumors of cancer patients: Initial clinical experience. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2020. doi:10.1158/1538-7445.AM2015-2020

e22177 Background: Cancer drug developers face a fundamental challenge in the way new candidate drugs are evaluated. Early stage compounds are tested in preclinical models that lack important compo...

Assessment of anti-cancer drug efficacy is an imprecise and challenging undertaking. Early candidate selection is typically based on results from systemically treated animal models and later by performance in human trials where patients are exposed to often toxic levels of drug, prior to obtaining readouts of tumor response. In both of these testing models, only one drug can be tested at a time. Using these methods, over 90% of candidate new oncology drugs fail to provide benefit for patients in human clinical trials. To improve the predictive value of preclinical candidate selection in animal models and enable a new type of pre-Phase 1 toxicity-sparing comparative drug efficacy study in humans, amenable for use in the solid tumor clinic, we have developed a technology platform called CIVO™. This platform allows for simultaneous assessment of multiple drugs or drug combinations directly in a single solid tumor to assess efficacy, resistance and drug synergies. In this study, precise, controlled delivery of classic chemotherapy drugs vincristine and doxorubicin induced spatially defined (ranging 0.3 – 2.0 mm in diameter), readily detectable, and mechanism-specific cellular changes around sites of tumor microinjection across three xenograft models of lymphoma. The extent of apoptosis induced via CIVO™ microdosing of each drug ( The data presented here generated in drug-responsive and non-responsive solid tumors in the preclinical setting sets the stage for future application of this technology to demonstrate tumor responsiveness to novel drug candidates in the context of human patients. Citation Format: Richard Klinghoffer, Alicia Moreno-Gonzalez, Michael Carleton, Marc Grenley, Beryl Hatton, Jason Frazier, William Kerwin, Ilona Tretyak, Nathan Hedin, Joyoti Dey, Joseph Casalini, Sally Ditzler, James Olson, Nathan Caffo. A platform to assess multiple therapy options simultaneously in a patient's own tumor. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A39.

Proper selection of anti-cancer agents at the earliest stage of patient treatment following diagnosis of disease relapse is expected to substantially impact clinical response to treatment. Currently, genomic approaches to personalized cancer treatments have been yielded mixed results, while empirical tests to assess tumor responsiveness have been limited to ex vivo systems that disrupt the native tumor microenvironment and show limited predictive value. To address the need for multiplexed in vivo chemosensitivity testing, we have developed a technology that allows simultaneous assessment of multiple cancer therapeutics directly in a patient's tumor. This technology could provide a valuable decision-making tool to prioritize effective treatments in the oncology clinic. Data herein highlight how this technology enables controlled and reliable microinjection of multiple drugs simultaneously in preclinical tumor models, canine lymphoma, and human lymphoma patients. Consistent with the controlled drug delivery of this system, spatially localized, readily detectable, and mechanism-specific cellular changes were observed around sites of microinjection in response to classic chemotherapy drugs (vincristine and doxorubicin) as well as to a small molecule inhibitor of TOR kinase. Importantly, localized response (or lack thereof) to individual components of CHOP combination therapy correlated with response to long-term systemic drug administration across multiple cell line and patient-derived xenograft models of lymphoma. Underscoring the importance of assessing drug efficacy in the context of an intact in vivo system, tumor responses to vincristine were impacted by the local tumor microenvironment. Our results also emphasize the importance of selecting effective therapies early in the course of treatment, as drug resistance mechanisms induced cross-resistance to otherwise efficacious drugs. These studies set the stage for use of this platform in oncology drug development, where the ability to more rapidly assess drug efficacy using clinically relevant in vivo tumors may decrease the current reliance on in vitro cell-based models of cancer and possibly increase the likelihood of clinical success. This platform may thus be useful a clinical decision-making tool for selection of patient-specific anti-cancer therapies. Citation Format: Richard Klinghoffer, Alicia Moreno-Gonzalez, Michael Carleton, Jason Frazier, Marc Grenley, Ilona Tretyak, Nathan Hedin, Joyoti Dey, Joseph Casalini, Beryl Hatton, Sally Ditzler, James Olson, Daniel Pierce, Ellen Filvaroff, Nathan Caffo. A platform to assess multiple therapy options simultaneously in a patient's own tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3129. doi:10.1158/1538-7445.AM2014-3129