ONCOLOGY LETTERS 15: 3061-3067, 2018 High expression of carcinoembryonic antigen and telomerase reverse transcriptase in circulating tumor cells is associated with poor clinical response to the immune checkpoint inhibitor nivolumab HALIN BAO1*, TUYA BAI1*, KOJI TAKATA2*, TAKEHIKO YOKOBORI1,3, TAKASHI OHNAGA2, TAKESHI HISADA4, TOSHITAKA MAENO4, PINJIE BAO1, TOMONORI YOSHIDA1, YUJI KUMAKURA1, HIROAKI HONJO1, MAKOTO SAKAI1, MAKOTO SOHDA1, MINORU FUKUCHI1, BOLAG ALTAN5, TADASHI HANDA6, MUNENORI IDE6, TATSUYA MIYAZAKI1, KYOICHI OGATA1, TETSUNARI OYAMA6, KIMIHIRO SHIMIZU7, AKIRA MOGI7, TAKAYUKI ASAO8, KEN SHIRABE7,9,10, HIROYUKI KUWANO1,7 and KYOICHI KAIRA5 1Department of General Surgical Science, Graduate School of Medicine, Gunma University, Maebashi, Gunma 371‑8511; 2Toyama Industrial Technology Center, Takaoka, Toyama 933‑0981; 3Research Program for Omics‑based Medical Science, Division of Integrated Oncology Research, Gunma University Initiative for Advanced Research; Departments of 4Respiratory Medicine, 5Oncology Clinical Development and 6Diagnostic Pathology, Graduate School of Medicine, Gunma University, Maebashi, Gunma 371‑8511; 7Integrative Center of General Surgery, Gunma University Hospital, Maebashi, Gunma, 371‑8510; 8Big Data Center for Integrative Analysis, Gunma University Initiative for Advance Research; 9Department of Hepatobiliary and Pancreatic Surgery, Graduate School of Medicine, Gunma University, Maebashi, Gunma 371‑8511, Japan Received July 10, 2017; Accepted December 13, 2017 DOI: 10.3892/ol.2017.7671 Abstract. The present study aimed to enrich circulating tumor large cell fraction had a high expression of CEA and hTERT, cells (CTCs) from blood samples using a new size-sorting CTC with the former being significantly associated with the clinical chip. The present study also set out to identify a blood sensi- response to nivolumab. The expression of CEA and hTERT in tivity marker for the immune checkpoint inhibitor nivolumab CTCs derived from the blood of a patient with lung cancer were in patients with advanced, pre-treatment lung cancer. The CTC also validated. The evaluation of CEA and possibly hTERT in sorting efficacy of the chip was investigated and the large cell CTCs collected by the CTC chip may represent a promising fraction of blood samples from 15 patients with pre-treatment predictive blood marker for sensitivity to nivolumab. To the lung cancer who were later administered nivolumab were puri- best of our knowledge this is the first report to describe the fied. The expression levels of carcinoembryonic antigen (CEA), predictive CTC marker for nivolumab in pre-treatment patients. human Telomerase Reverse Transcriptase (hTERT), cyto- keratin19 (CK19), and programmed death ligand-1 (PD‑L1) Introduction were investigated to clarify the association between these CTC markers and the clinical response to nivolumab. The CTC chip Immune checkpoint inhibitors have recently attracted effectively enriched cells from lung cancer cell line PC-9. The attention as an innovative cancer therapy (1). Programmed death 1 (PD-1)/programmed death ligand-1 (PD-L1) checkpoint inhibitors have been shown to have a continuous clinical effect and low toxicity in some responder patients with several types of recurrent and metastatic diseases (2). Correspondence to: Dr Takehiko Yokobori, Department of Among these, the anti PD‑1 monoclonal antibody nivolumab General Surgical Science, Graduate School of Medicine, Gunma has led to a good clinical response in several cancer patients University, 3‑39‑22 Showamachi, Maebashi, Gunma 371‑8511, Japan E-mail: [email protected] with lung cancer, melanoma, and renal cell carcinoma (3-6). However, many cancer patients do not gain sufficient benefits *Contributed equally even with the anti-PD-1 antibody treatment. Some researchers have proposed that the level of PD‑L1 expression and the DNA Key words: immune checkpoint inhibitor, circulating tumor cells, mismatch-repair status in a tumor are biomarker candidates for CTC chip, size sorting predicting the sensitivity to immune checkpoint blockade (3,7). However, invasive tumor sampling is required to determine the protein expression and DNA status in tumor cells. Therefore, 3062 BAO et al: BLOOD SENSITIVITY MARKER FOR NIVOLUMAB further research is needed globally to identify a new blood this study were as follows: pathologically proven lung cancer; biomarker that does not require invasive sampling. recurrent lung cancer candidate for nivolumab as a result of In cancer patients, circulating tumor cells (CTCs) in progressive disease (PD) following chemotherapy; Eastern peripheral blood have been identified as a reliable blood tumor Cooperative Oncology Group (ECOG) performance status marker (8,9). The evaluation of CTCs generally includes an of 0-2, 2-[18F]‑fluoro‑2‑deoxy‑D‑glucose 18( F‑FDG) positron enrichment step and detection processes depending on the emission tomography/computed tomography (PET/CT) sched- CTC characteristics, such as tumor size, density, and cell uled before and after the first cycle of nivolumab therapy; surface antigen expression, which are conducted using a no evidence of concurrent cancer; no uncontrolled diabetes cytometric-based or polymerase chain reaction (PCR)-based mellitus; no interstitial pneumonia or pulmonary fibrosis; and method (10). Nagrath et al (11), were the first to report on a adequate organ function. new microfluid device known as a CTC chip that was coated A pre‑treatment 18F‑FDG‑PET/CT study and blood with epithelial cell adhesion molecule (EpCAM) antibody, sampling for CTC sorting were performed as part of the which could be used to collect CTC‑expressing EpCAM disease evaluation workup prior to the administration of from whole blood samples of cancer patients. Because nivolumab. If the tumor size was successfully suppressed CTCs are known to be larger than nearly all normal blood by 3 months after the initiation of nivolumab, a subsequent cells (12,13), the use of only the large cells in blood samples post-treatment PET/CT was considered at around this time as PCR templates will enable CTCs to be detected more easily at the discretion of the investigator. The clinical response using a highly sensitive PCR-based method against the CTC to nivolumab was assessed from the 18F‑FDG uptake and markers carcinoembryonic antigen (CEA), human Telomerase chest CT of these patients, and categorized as a partial Reverse Transcriptase (hTERT), cytokeratin 19 (CK19), and response (PR), stable disease (SD), or PD according to the PD‑L1 (9,14-16). Therefore, in the present study, we used a Response Evaluation Criteria in Solid Tumors (RECIST). modified CTC chip that was based on a continuous particle All blood samples were prospectively collected and used in separation method (17) to enrich CTCs according to cell size. accordance with the Helsinki Declaration and the guidelines The purpose of the present study was to utilize this new of the Gunma University Ethical Review Board for Medical CTC chip to collect the large cell fraction from whole blood Research Involving Human Subjects after obtaining written samples and to find a blood sensitivity marker for nivolumab informed consent from each patient (approval no. 1404). in advanced, pre-treatment lung cancer patients. To do this, The human lung cancer cell line PC-9 and breast we first examined the sorting efficacy of the new CTC chip cancer cell lines MCF7 and MDA‑MB‑231 were used to using the lung cancer cell line PC-9 and then evaluated the examine the efficacy of the CTC chip for sorting cancer mRNA expression of the CTC markers CEA, hTERT, CK19, cells according to size. These cell lines were provided by and PD‑L1 in the large cell fraction of clinical lung cancer the RIKEN BioResource Center and the American Type patients' blood samples collected by this chip. Culture Collection. The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with Materials and methods 100 units/ml penicillin, 100 units/ml streptomycin, and 10% fetal bovine serum (FBS) in a humidified 5%‑CO2 incubator Preparation of a polymeric CTC chip for size sorting. A poly- at 37˚C. Before cell sorting, these cells were labeled using meric CTC chip for size sorting was produced as previously the CellTrace™ Carboxyfluorescein Diacetate Succinimidyl described (18). The chip was set in a holder to enable liquid Ester (CFSE) Cell Proliferation kit (Thermo Fisher Scientific, samples to flow from two inlets to two outlets. Cell suspen- Inc., Waltham, MA, USA), according to the manufacturer's sion samples and mere buffer were sent from each inlet tube protocol. The labeled cells were then spiked into the CTC chip into the CTC chip at 0.2 ml/min using a syringe pump (Fig. 1). and separated into the small or large cell fraction (Fig. 2A), Two outlets were used to allow the large cell fraction including following which the sorted cancer cells in each fraction were CTCs to be enriched and blood cells in the small cell frac- counted microscopically. tion to be collected separately. Since the diagonally-arranged microposts cannot influence the flow of small cells, these are Reverse transcription‑polymerase chain reaction (RT‑PCR). To carried into the small cell fraction simply as a result of the discover a useful biomarker for predicting patients' sensitivity to buffer flow. By contrast, large cells, which include CTCs, are the immune checkpoint inhibitor nivolumab, we examined the sorted by the microposts based on their size (Fig. 1B and C). relationships between