Abstract 2223: Analytical validation of a novel circulating tumor cell platform (CMx) for early detection of cancer
Authors: Shao Hung-Jen , Hsieh Huangpin B. , Tsai Wen-Sy , Wu Jen-chia , Lai Jr-Ming , Chang Shih-En , Javey Mana , Segurado Oscar , Amin Mahul B. , Nimgaonkar Ashish , Mei Rui 
Published: August 2018

Abstract
Introduction Circulating Tumor Cells (CTCs) are shed into the blood early by in situ neoplasm and are the mechanism by which cancer metastasizes. CTCs are generally precursors to ctDNA and can be used for early cancer detection. However, to the best of our knowledge, no platform has been analytically validated to detect CTCs in early stage/pre-cancer when these cells are present at a very low concentration in blood.
Methods The CellMax CTC (CMx) biomimetic platform uses a proprietary surface modified microfluidic biochip that sensitively captures CTCs. The analytical performance of the test was established by spiking low EpCAM-expressing colorectal cancer cell line HCT116 into 2mLs of healthy donor blood to achieve three different concentrations, with ~3 cells as “low”, ~12 cells as “medium” and ~96 cells as “high” concentrations. Cytokeratin 20, a more specific epithelial cell marker than pan-cytokeratin, and CD45, a WBC marker were used for staining.
Results Overall efficiency (O/E, intact cells recovered vs. input) for 40 spiked donor samples was 34% (4.6% standard deviation (SD) and 13.6% CV) and met the requirements for College of American Pathologists accreditation. No CTCs were detected in 5 unspiked controls, confirming 100% analytical specificity. Assay limit of detection (sensitivity) was a low 3-cells-per-2mL-blood and shown to be linear across all concentrations. The linear regression equation slope=0.23, intercept=0.82 and R2=0.97, demonstrating that the detection of CTCs was linear over the reportable range (3 to 129 CTCs per 2 mL of blood). The precision (reproducibility) of the assay as assessed by using triplicate samples at 3 levels of concentrations over 7 days, for 63 samples, was 36.9% O/E (Low, SD=9.2%), 34.2% (Medium, SD=12.1%), and 31% (High, SD=6%). The highest SD of 12.1% compares favorably to the SD for the CellSearch Assay (18%) in their publicly available 510K filing. In 398 routine quality control samples with cell lines spiked into growth medium, which better reflects process variability than blood variability, the mean O/E was 66.7%, SD was 9.9% and CV was 14.9%. Clinical proof for early detection was established under IRB by testing 170 patients with colorectal diseases ranging from pre-cancerous lesions to stage I-IV cancer: 28% (N=48) were colonoscopy-negative, 72% (N=142) had diagnostically confirmed colorectal neoplasms or cancer. Detection sensitivity for precancerous lesions, including adenomatous and dysplastic polyps, was 47% (with 94% specificity), which is higher than the sensitivity for the existing stool-based standard FIT (24%), and Cologuard (42%) per the latter's publicly available FDA submission.
Conclusion The analytical validation of CMx CTC platform showed better precision than the FDA-approved CellSearch and the clinical study confirmed higher sensitivity for the detection of pre-malignant colorectal cancer than existing stool-based tests.
Citation Format: Hung-Jen Shao, Huangpin B. Hsieh, Wen-Sy Tsai, Jen-chia Wu, Jr-Ming Lai, Shih-En Chang, Mana Javey, Oscar Segurado, Mahul B. Amin, Ashish Nimgaonkar, Rui Mei. Analytical validation of a novel circulating tumor cell platform (CMx) for early detection of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2223.