Detection of circulating tumor cells in drainage venous blood from colorectal cancer patients using a new filtration and cytology-based automated platform.

Masayuki Tsutsuyama,Masayuki Tsutsuyama,Mayumi Yoshimura,Kentaro Wajima,Koji Komori,Seiji Ito,Taihei Oshiro,Seichin Kinuta,Yasushi Yatabe,Yasuhiro Shimizu,Yasufumi Sakakibara,Yasuhiro Kodera,Takashi Kinoshita,Yoshiyuki Ichinosawa,Hayao Nakanishi

doi:10.1371/journal.pone.0212221

Masayuki Tsutsuyama, Masayuki Tsutsuyama + Show 13 more

Open Access

https://doi.org/10.1371/journal.pone.0212221

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Journal: PloS one	Publication Date: Feb 27, 2019
Citations: 17	License type: CC BY 4.0

Affiliation: Aichi Cancer Center, Nagoya University

Abstract

Numerous technologies exist to detect circulating tumor cells (CTCs), although reports on cytological detection of CTCs remain limited. We recently developed a cytology-based CTC detection device using glass slides and light microscopy. In this study, we automated this previously manual device to improve its efficiency and cost effectiveness for clinical applications. We conducted a pilot study using this device to compare CTCs in peripheral blood (PB) and draining venous blood (DVB) from patients with colorectal cancer (CRC). The cytology-based automated CTC detection platform consisted of a disposable filtration device with a three-dimensional (3D) metal filter and multichannel automated CTC enrichment device. This platform allowed rapid and gentle filtration of CTCs and their efficient transfer from the filter to glass slides for subsequent Papanicolaou (Pap) and immunocytochemical (ICC) staining. Cytological diagnosis of CTCs was performed by observing permanent glass slide specimens by light microscopy. The current pilot clinical study enrolled CRC patients (n = 26) with stage I–IV tumors, who underwent surgery. PB was collected before surgery, and DVB was obtained from the mesenteric vein immediately after resection. Based on the CTC morphology obtained from PB and DVB samples, we proposed the following cytological criteria for the diagnosis of CTCs: pan-cytokeratin-positive, atypical cells with malignant morphological features identified by Pap staining. The numbers of CTCs defined by these criteria were significantly higher in DVB than PB from CRC patients (p<0.01), and the number of CTCs in DVB was increased significantly with stage progression (p<0.05). These results suggest that DVB may be another potential source of CTCs other than PB for liquid biopsies including downstream analysis. This automated cytology-based CTC detection device therefore provides a unique and powerful tool to investigate the significance of CTCs in CRC patients in a clinical setting.

Highlights

Colorectal cancer (CRC)-related mortality has decreased over the past two decades as a result of advances in treatment modalities such as pre- and post-operative chemotherapies in combination with antibody-based therapies
Detection of Circulating tumor cells (CTCs) in peripheral blood (PB) and draining venous blood (DVB) from CRC patients. Using this CTC enrichment platform and cytological criteria for CTCs, we evaluated the feasibility of the CTC detection system using PB and DVB from 26 CRC patients (Table 1) and PB from 14 healthy volunteers
We improved on our previous device [13] and developed a new automated cytology-based, multichannel CTC detection platform with several unique characteristics

Summary

Introduction

Colorectal cancer (CRC)-related mortality has decreased over the past two decades as a result of advances in treatment modalities such as pre- and post-operative chemotherapies in combination with antibody-based therapies. Mortality due to metastatic CRC has remained essentially unchanged in the past decade and is still the most common cause of CRC-related death [1] Conventional diagnostic techniques, such as serum tumor markers and computed tomography/positron emission tomography, are usually ineffective for early diagnosis of metastasis and to evaluate the risk of postoperative metastatic recurrence in CRC patients [2]. Cell-free circulating tumor DNA has recently emerged as a new form of liquid biopsy to detect genetic alterations in patients with advanced stage CRC, its use as an early diagnostic marker for metastasis is still limited [3]. CTCs have usually been identified by immunophenotypical criteria such as keratin+/EpCAM+/CD45−/DAPI+ as judged by dark field immunofluorescence (IF) [9] This approach cannot provide sufficient morphological and cytological information on CTCs and their background cellular constituents in blood. CTC detection platforms based on epitope-dependent IF criteria have been associated with the risk of missing CTCs with the epithelial-mesenchymal transition (EMT) phenotype [10]

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