Abstract 1734: Absolute quantification of circulating tumor cell RNA enables high specificity detection of hepatocellular carcinoma
Authors: Kalinich Mark , Bhan Irun , Kwan Tanya T. , Miyamoto David T. , Javaid Sarah , LiCausi Joseph A. , Milner John D. , Hong Xin , Goyal Lipika , Sil Srinjoy , Choz Melissa , Kapur Ravi , Muzikansky Alona , Zhang Huidan , Weitz David A. , Sequist Lecia V. , Ryan David P. , Chung Raymond , Zhu Andrew X. , Isselbacher Kurt J. , Ting David T. , Toner Mehmet , Maheswaran Shyamala , Haber Daniel A. 
Published: July 2017

Abstract
Background: Although liver cancer has the second-highest mortality rate among cancers internationally, accurate and scalable assays for the early detection and longitudinal monitoring of hepatocellular carcinoma are lacking. Circulating tumor cells are released from invasive cancers into the blood stream, but the difficulty inherent in isolating, identifying, and characterizing these ultra-rare cells has precluded their widespread implementation as a biomarker. By combining a high-throughput microfluidic negative depletion CTC isolation strategy with the absolute quantification of lineage-specific RNAs, we report the highly specific detection of hepatocellular carcinoma CTCs from patient blood draws.
Methods: Blood draws from 48 hepatocellular carcinoma patients, 31 chronic liver disease patients, 25 healthy donors, and 44 patients with primary cancers other than hepatocellular carcinoma were processed through the microfluidic device (CTC-iChip). RNA was extracted, whole-transcriptome amplified, and quantified using droplet digital PCR. Transcript counts were used to fit a logistic regression model to integrate distinct transcript levels into a single CTC-score. The technical feasibility of utilizing RNA sequencing for identification of novel CTC transcripts of interest was also demonstrated with a liver cancer cell line spike-in study.
Results: 9 of the 16 untreated HCC patients were successfully detected, while only 1/31 chronic liver disease patients were incorrectly classified. HCC patients undergoing treatment showed a significant decrease in their CTC-score; only 9/32 patients actively receiving treatment were positive. The CTC-score was not correlated with the HCC serum biomarker alpha-fetoprotein, and combining these two orthogonal measures led to estimated positive and negative predictive values of 80% and 86%, respectively, in a high-risk cohort. RNAseq analysis of cell line spike-in data revealed the potential of RNA sequencing for uncovering novel transcripts of interest.
Conclusion: Coupling microfluidic depletion with droplet digital PCR allows for the highly specific detection of hepatocellular carcinoma. The CTC-score generated from these data tracks with clinical intervention and is orthogonal to the existing biomarker AFP; combining these two assays has the potential to provide superior detection compared to either individual approach.
Citation Format: Mark Kalinich, Irun Bhan, Tanya T. Kwan, David T. Miyamoto, Sarah Javaid, Joseph A. LiCausi, John D. Milner, Xin Hong, Lipika Goyal, Srinjoy Sil, Melissa Choz, Ravi Kapur, Alona Muzikansky, Huidan Zhang, David A. Weitz, Lecia V. Sequist, David P. Ryan, Raymond Chung, Andrew X. Zhu, Kurt J. Isselbacher, David T. Ting, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Absolute quantification of circulating tumor cell RNA enables high specificity detection of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1734. doi:10.1158/1538-7445.AM2017-1734