非小细胞肺癌患者的环肿瘤细胞分析

Analysis of Circulating Tumor Cells in Patients with Non-small Cell Lung Cancer Using Epithelial Marker-Dependent and -Independent Approaches

Introduction: Epithelial circulating tumor cells (CTCs) are detectable in patients with non-small cell lung cancer (NSCLC). However,epithelial to mesenchymal transition, a widely reported prerequisite for metastasis, may lead to an underestimation of CTC number. We compared directly an epithelial marker-dependent (CellSearch) and a marker-independent (isolation by size of epithelial tumor cells [ISET]) technology platform for the ability to identify CTCs. Molecular characteristics of CTCs were also explored.

Methods: Paired peripheral blood samples were collected from 40 chemona¨ive, stages IIIA to IV NSCLC patients. CTCs were enumerated by Epithelial Cell Adhesion Molecule-based immunomagnetic capture (CellSearch, Veridex) and by filtration (ISET, RareCell Diagnostics). CTCs isolated by filtration were assessed by immunohistochemistry for epithelial marker expression (cytokeratins,Epithelial Cell Adhesion Molecule, epidermal growth factor receptor) and for proliferation status (Ki67).

Results: CTCs were detected using ISET in 32 of 40 (80%) patients compared with 9 of 40 (23%) patients using CellSearch. A subpopulation of CTCs isolated by ISET did not express epithelial markers.Circulating tumor microemboli (CTM, clusters of
3 CTCs) were observed in 43% patients using ISET but were undetectable by CellSearch. Up to 62% of single CTCs were positive for the proliferation marker Ki67, whereas cells within CTM were nonproliferative.

Conclusions: Both technology platforms detected NSCLC CTCs.ISET detected higher numbers of CTCs including epithelial marker negative tumor cells. ISET also isolated CTM and permitted molecular characterization. Combined with our previous CellSearch data confirming CTC number as an independent prognostic biomarker for NSCLC, we propose that this complementary dual technology approach to CTC analysis allows more complete exploration of CTCs in patients with NSCLC.

The detection and characterization of circulating tumor cells (CTCs) in cancer patients offer as yet untapped potential to further understand the biology of human cancer metastasis and to identify novel treatment strategies. Recent advances in technology have paved the way to reproducible CTC detection and enumeration and begun to reveal their potential as a real-time, minimally invasive, “virtual biopsy”(reviewed in Hou et al.1). The robust, semiautomated Cell-Search platform (Veridex, LLC, Raritan, NJ) has been used to demonstrate prognostic significance of CTC numbers in patients with metastatic breast, prostate, and colorectal cancers2–7 (reviewed by Krebs et al.8) and subsequently has been approved by the US Food and Drug Administration as a prognostic biomarker and as an aid to monitoring treatment response in these disease types. We recently reported that CellSearch detection of
5 CTCs (per 7.5 ml of blood) in patients with advanced non-small cell lung cancer (NSCLC) is a poor prognostic factor and that a change in CTC number after a single cycle of standard-of-care chemotherapy predicts survival outcome.9 However, two-thirds of patients with stage IV NSCLC had no detectable CTCs, and CTCs were detectable in less than 5% stage III patients using this technology platform.

Detection of CTCs using CellSearch is dependent on tumor cell expression of Epithelial Cell Adhesion Molecule(EpCam), an epithelial cell marker. However, the paradigm of epithelial to mesenchymal transition (EMT) as a predominant mechanism for tumor cell invasion and metastasis raises the possibility that not all cells in the circulation will express epithelial markers.10–14 Thus, we hypothesized that the low prevalence of CTCs detected with the CellSearch technology in patients with advanced NSCLC, although strongly prognostic,may be due to the loss of EpCam expression. Therefore,we explored an antigen/EpCAM-independent approach for the ability to identify CTCs and for the potential to perform molecular characterization.

FIGURE 1. Biological controls for immunohistochemistry of circulating tumor cells (CTCs) isolated by ISET. (A)Positive-staining controls and (B) negative-staining controls for each of the markers used in this study to characterize CTCs/CTM. Control staining was performed on cell lines or peripheral blood mononuclear cells (PBMCs),cytospun onto glass slides. All images were acquired at 400 magnification using a Bioview imaging system.ISET, isolation by size of epithelial tumor cells; CTM, circulating tumor microemboli.

FIGURE 2. Exclusion of nontumor cell contaminants on  ISET filters and circulating tumor cell (CTC) enumeration strategy. A,CD45 immunostaining of H460 tumor cells spiked into healthy donor blood and processed through the ISET filtration system to exclude white blood cells. Filter pores appear dark and circular or cylindrical (black arrows), white blood cells stain brown due to DAB-substrate reaction (red arrow), and tumor cells appear blue due to the hematoxylin counterstain (blue arrow). B,Bar chart demonstrating the mean and SE for CTC number depending on the number of spots analyzed. Four spots or more exhibited the most representative mean CTC value. C, VE-cadherin (CD144) staining on clinical samples processed by ISET to exclude mature circulating endothelial cells (CECs). CECs exhibited small, round, or oval, pale nuclei with low nuclear-to-cytoplasmic ratio and were positive for CD144 (blue arrows). Nuclei appear blue due to hematoxylin counterstain. A cluster of CECs is shown in the central panel. Filter pores appear dark and circular (black arrows). D, CD45 staining on clinical samples processed by ISET. Cells with CEC morphology are seen with hematoxylin-stained nuclei (blue arrows). These cells were considered to be mature CECs on the basis on their morphological similarity with CD144-positive cells (C) and absence of CD45 expression. E, Typical CD45 negative squamous skin cells exhibiting small, round, pyknotic nuclei with abundant cytoplasm
(blue arrows). Filter pores appear dark and circular (exemplified by black arrow). ISET, isolation by size of epithelial tumor cells.