Abstract 576: Development and analytical validation of a novel assay for tissue detection of hyaluronan in the tumor microenvironment to select patients for molecularly targeted pancreatic cancer therapies
Authors: Gelb Arnold B. , Jiang Ping , Jadin Laurence , Maneval Daniel C. , Shepard H. Michael 
Published: August 2015

Abstract
Background: Hyaluronan (HA), a glycosaminoglycan distributed in the extracellular matrix, is frequently upregulated in cancer and correlates with disease progression. Administration of a first-in-class PEGylated recombinant human hyaluronidase (PEGPH20) depletes HA, unmasks HA around tumor cells and decreases tumor interstitial fluid pressure, increasing accessibility to systemically administered agents in preclinical models. PEGPH20 recently received Orphan Drug designation in pancreatic ductal adenocarcinoma (PDA) in the USA. We report the development and analytical validation of a novel prototype assay for a companion diagnostic to select patients with PDA for molecularly targeted therapies with PEGPH20.
Design: A recently described biotinylated recombinant immunoadhesin (HTI-601, Jadin 2014) was adapted for use in an immunohistochemistry-based assay on formalin-fixed paraffin-embedded tissue. Sensitivity, specificity, and within-laboratory precision studies were performed in a research mode and then at a central laboratory on a validation set of approximately 200 tumor and normal tissues under GCP conditions. Both pathologist scoring and operator-assisted image analysis (positive pixel count for strong positive pixels) were evaluated.
Results: Analytical sensitivity studies identified an optimal probe dilution of 0.417 μg/mL on an immunostainer based on dynamic range in 4 human tumor xenografts containing differential levels of HA. The frequency of high HA observed was 62.7% of archival PDA (N = 75). During analytical specificity, 5.1% of 75 PDA and 3 normal adjacent tissue samples had faint or focal staining after pre-digestion with recombinant PH20 hyaluronidase. No cross-reactivity was identified in 99 normal human tissues across a panel of histotypes; only 1 (1.0%) colon sample had staining of macrophages. Within-laboratory precision among three observers’ annotations had average%CVs as follows: between day 4.8%, between run 12.2%, and repeatability 16.6%.
Conclusion: The sensitivity, specificity and within-laboratory precision of the prototype assay using image analysis are acceptable. The frequency of high HA expression is similar to other studies in the literature. Cross-reactivity was focal and faint so is unlikely to interfere. Subset analysis showed contributions to%CV to be higher for HAlow than HAmedium or HAhigh samples as expected. These findings warrant further evaluation of HTI-601 staining on biopsies for PEGPH20-associated therapies in clinical studies.
Citation Format: Arnold B. Gelb, Ping Jiang, Laurence Jadin, Daniel C. Maneval, H. Michael Shepard. Development and analytical validation of a novel assay for tissue detection of hyaluronan in the tumor microenvironment to select patients for molecularly targeted pancreatic cancer therapies. [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 576. doi:10.1158/1538-7445.AM2015-576