Abstract 2186: Validation of a 32-gene classifier for the subtyping of carcinomas using the qNPA™ ArrayPlate Platform
Authors: Omolo Bernard , Centeno Barbara , Yeatman Timothy 
Published: August 2015

Abstract
Introduction: The majority of adult malignancies are carcinomas which are broadly separated into four types: adenocarcinomas, squamous cell carcinomas, neuroendocrine carcinomas and urothelial carcinomas. These can usually be differentiated using routine morphology and immunohistochemistry, but occasionally this distinction is difficult. We developed a 32-gene classifier using microarray analysis of frozen tissue, to differentiate among these four main types of epithelial malignancies. In this study, we sought to validate our 32-gene classifier on formalin fixed, paraffin embedded (FFPE) tissue using the qNPA™ ArrayPlate Platform by HTG.
Materials and methods: A total of 288 FFPE primary tumors were retrieved the Moffitt Cancer Center Anatomic Pathology Department. Samples were analyzed using a custom 96 well micro titer qNPA array plates supplied by the manufacturer. The qNPA assays were carried out following the manufacturer's protocol (HTG, Tucson, AZ). Digital images of the array plates were analyzed using HTG's SuperCapella Imaging System software. The samples were used in developing models utilizing gene expression profiles to predict the class (tumor type) of future samples, based on the nearest shrunken centroids algorithm and implemented using the Prediction Analysis of Microarrays (PAM) tool in BRB-ArrayTools. We estimated the prediction error of the models using 10-fold cross-validation. For each 10-fold training set of 193 samples, the entire model building process was repeated, including the gene selection process.
Results: The classifier had a 13.7% error rate overall. The sensitivity, specificity, positive predictive value and the negative predictive value for each tumor type were as follows (%): 93.8, 85, 81.5, 95 for adenocarcinoma; 82.1, 97.6, 85.2, 97 for neuroendocrine carcinomas, 89.1, 92.2, 85.1, 94.4%for squamous cell carcinoma and 33.3, 100, 100 and 92.5 for urothelial carcinomas.
Conclusions: The assay had a high sensitivity for the identification of adenocarcinomas, squamous cell carcinomas and neuroendocrine carcinomas, but lacked sensitivity for the identification of urothelial carcinomas. Urothelial carcinomas were misclassified as either squamous cell carcinomas or adenocarcinomas. Similar difficulties are encountered with immunohistochemistry, due to the overlap in protein expression between squamous cell carcinoma and urothelial carcinoma. GATA3, one of the genes used to classify urothelial carcinomas, is also expressed by breast carcinomas, which could account for the difficulty in differentiating urothelial from adenocarcinomas.
Key words: carcinoma; class prediction; microarray; validation
Citation Format: Bernard Omolo, Barbara Centeno, Timothy Yeatman. Validation of a 32-gene classifier for the subtyping of carcinomas using the qNPA™ ArrayPlate Platform. [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 2186. doi:10.1158/1538-7445.AM2015-2186