Condjella, Rachel

Abstract Eosinophil infiltration into tumors has been associated with positive and negative growth human cancers and in mouse models of tumorigenesis. Our previous studies have shown that eosinophils infiltrate tumors and differentially accumulate in the necrotic and capsule regions of tumors. Using various eosinophil-deficient mouse models, we demonstrate that in the absence of eosinophils tumor growth is attenuated. Methods: Tumor growth kinetics were assessed using a B16F10 melanoma subcutaneous cell injection model. Eosinophil chemotaxis was assessed using an ex vivo transwell insert assay system. We compared tumor growth in wild type animals vs. various eosinophil-deficient mouse models that are transgenic line of mice (PHIL) and MBP-/-/EPO-/- double knockout mice that are deficient in eosinophil-specific proteins (major basic protein (MBP) and eosinophil peroxide (EPO)) resulting in a blockade of eosinophilpoiesis. Conclusions: The migration of eosinophils was inhibited through blocking the sphingosine-1-phosphate pathway, suggesting recruitment and accumulation is primarily an inflammatory response. Tumor growth was decreased by ~40-60% in PHIL and in MBP-/-/EPO-/- double knockout mice relative to tumors in wild type animals. In addition, EPO-/- mice, and not MBP-/-mice, replicate this ~40% reduction in tumor size implicating a significant role for EPO in tumor growth kinetics. These data suggest eosinophils respond to inflammatory mediators and mediate augment tumor growth.

Abstract Atopic dermatitis and allergic contact dermatitis are two of the most common skin diseases. In both diseases, eosinophils have been suggested as potential contributors of the pathologies linked with these skin conditions. Our goal was to establish a causative relationship between the inflammatory response of chemically induced contact hypersensitivity and eosinophil-mediated activities. Wild type and eosinophil-less PHIL mice were sensitized and challenged with 2,4-dinitrochlorobenzene (DNFB) or alternatively trimellitic anhydride (TMA). Time lapsed videography was used to capture the number of itching events associated with treatment. Immunohistochemistry for eosinophils and nerves was performed to correlate the effect of dermatitis on innervation and eosinophil accumulation in these mice. Our studies showed that in both the DNFB and TMA models a robust eosinophil infiltrate occurred that was accompanied by copious levels of eosinophil degranulation. More importantly, these studies demonstrated that metrics of inflammation (e.g., induced ear swelling (i.e., thickness)) was reduced in PHIL mice relative to wild type. Our observations were also directly correlative with a loss of nerve growth and branching in PHIL mice. These data implicate eosinophils as contributors to the pathologies linked with chemically-induced contact hypersensitivity, including the induced itch responses associated with these models.

463 Background: The genomic assessment of cancer has been revolutionized by next-generation sequencing and is increasingly being applied in the clinic to guide therapeutic decision-making. Time to reporting of results, specimen quantity, and analyte quality have constrained initial clinical applications to gene panels and whole exome based strategies. Methods: Patients underwent surgical resection, excisional or core biopsies, or bone marrow biopsy. Samples were analyzed by whole genome or exome sequencing and RNA sequencing, bioinformatics analysis, and therapeutic target prioritization by a multi-disciplinary Clinical Genomics Board. All prioritized targets were CLIA validated using Sanger sequencing, RT-qPCR, FISH, or IHC as appropriate. Treatment was delivered using off-label FDA approved drugs, clinical trials, or single patient INDs. Results: We have enrolled 40 patients with advanced, treatment-refractory cancers of whom sequencing data is available on 33. The initial 6 patients were evaluated in a non-CLIA pilot phase and 27 in the CLIA enabled phase. Upon availability of the initial report, identified targets of putative therapeutic relevance were then prioritized by the CGB in 22/27 patients (81%) for subsequent CLIA validation. Eleven patients have been treated with genomically selected therapy with partial response in 3/10 assessed patients. A testicular cancer patient had aberrations in two testes specific genes, a copy number gain in TSSK6 and a novel gene fusion between thyroid hormone receptor associated protein 3 (THRAP3) and Tektin 2 (TEKT2). Additionally, a case of papillary renal cell carcinoma had an amplification of YAP1 and a previously unreported P287T mutation in CCND1, suggesting potential benefit with a CDK4/6 inhibitor. Treatment is ongoing and results will be reported. Conclusions: Integrated whole genome analysis in a CLIA setting is feasible. Integration of SNV, copy number and transcriptional data may allow for selection of putative driver genes to enhance targeted therapy decisions. Barriers for future broader implementation include the need for reduced time from biopsy to report and availability of therapies.

Abstract Background: Whole genome/exome/RNA sequencing has revolutionized the ability to assess the genomic landscape of cancer and is increasingly being utilized for clinical decision-making. Initial clinical applications have been constrained by specimen quantity, analyte quality and the time from sample acquisition to results report. Methods: Patients with advanced cancers underwent surgical resection, excisional/core biopsies, or bone marrow biopsy. Samples were analyzed by whole genome or exome sequencing in addition to RNA sequencing, bioinformatics analysis, and therapeutic target prioritization by a multi-disciplinary Clinical Genomics Board. All prioritized targets were CLIA validated using Sanger sequencing, RT-qPCR, FISH, or IHC as appropriate. Treatment was delivered using off-label FDA approved drugs, clinical trials, or single patient INDs. Results: We have enrolled 40 patients for whom sequencing data is available on 33. The initial 6 patients were evaluated in a non-CLIA pilot phase and 27 in a CLIA-enabled phase. Tumor types in the CLIA-enabled phase with the highest enrollment were pancreatic cancer (n=8) and cholangiocarcinoma (n=8). We sought to quantify the targets identified along with clinical benefit, defining these as the “Actionable Index” (AI) (proportion of patients with ≥ 1 putative drug target) and “Utility Index” (UI) (proportion of patients who derive clinical benefit). Putative therapeutic targets were identified in 7/8 (AI=0.88) cholangiocarcinoma (CC) patients and in 5/8 (AI=0.63) pancreatic cancer (PC) patients. All 3 CC patients who received target directed treatment achieved a partial response (UI=0.38). In contrast, none of the 4 PC patients who received target directed therapy had treatment response (UI=0.0). Interestingly no actionable targets were identified in 1 CC and in 2 PCs. One CC with an identified target was unable to access the drug and subsequently died. A CC patient and a PC patient, each with identified targets, expired prior to the initiation of therapy. Conclusions: While whole genome/exome/RNA sequencing is providing unparalleled detail of tumor genomes, the application to the clinic must be carefully considered. Actionability of targets will eventually need to be defined in close relation to eventual clinical utility and appropriate refinements to disease-gene-drug databases implemented. Preliminary observations in pancreatic cancer and cholangiocarcinoma demonstrate disparity in correlation between utility indices and actionable indices. Application of these tools in larger cohorts and types of tumors will need to be conducted to ascertain more precise estimates. Additional measures that are organ-site agnostic but pertain to specific targets (e.g. BRAF) will also need to be developed in order to facilitate more judicious application of sequencing in the clinical setting. Citation Format: Jan B. Egan, Alan H. Bryce, Mia D. Champion, Winnie S. Liang, Rafael Fonseca, Ann E. McCullough, Michael T. Barrett, Katherine Hunt, Rachel M. Condjella, Robert R. McWilliams, Stephen D. Mastrian, Janine LoBello, Daniel Von Hoff, David W. Craig, A. Keith Stewart, John D. Carpten, Mitesh J. Borad. Indices of actionability and clinical utility in a CLIA-enabled study of whole genome/exome/RNA sequencing in 33 cancer patients: Actionable vs. utility. [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 4694. doi:10.1158/1538-7445.AM2014-4694