Epithelial circulating tumor cells with a heterogeneous phenotype are associated with metastasis in NSCLC

Yujuan Zhang,Junling Li,Yu Men,Puyuan Xing,Jianyang Wang,Wei Cui,Jun Zhao,Danfei Xu,Zhouguang Hui

doi:10.1007/s00432-021-03681-9

Abstract

ObjectivesTo analyze the clinical relevance of heterogeneous phenotypes of peripheral circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC).Materials and MethodsCTCs in 5 mL venous blood were enriched using the Canpatrol™ CTC technique in 82 NSCLC patients. And then, CTCs were subjected to RNA in situ hybridization with a combination of epithelial (EpCAM and CK8/18/19) and mesenchymal (vimentin and TWIST1) markers.ResultsAccording to the fluorescent dots, CTCs were classified into three groups, including epithelial CTCs (E-CTC), hybrid epithelial/mesenchymal phenotypes (E/M-CTCs) and mesenchymal CTCs (M-CTCs). In 82 NSCLC cohort, only 2 patients didn’t detect CTCs, the overall CTCs detection rate was 97.5% (80/82). For 60 treatment naïve NSCLC, only one patient didn’t detect CTCs. The median number of total CTCs, hybrid E/M phenotype CTCs, E-CTCs and M-CTCs per 5 mL blood was 22 (range 1–90), 13 (range 0–83), 1 (range 0–17 and 0–47), respectively. Hybrid E/M CTCs, especially the e = m-CTCs, significantly differed between patients with and without distant metastasis. M-CTCs in advanced NSCLC patients were significantly more than the numbers observed in early stage patients. Patients with pure hybrid E/M-CTCs showed a lower proportion in distant metastasis positive cohort compared to negative ones (7% vs 22%), while patients with E + E/M CTCs (20% vs 9%) and E/M + M CTCs (33% vs 20%) showed a higher proportion. CTCs dynamic changes after treatment in 12 advanced NSCLC patients suggested that hybrid E/M-CTCs were related to the primary tumor size at baseline, while M-CTCs may suggest the progression of NSCLC.ConclusionWe concluded that E-CTCs with a hybrid E/M phenotype are associated to metastasis in therapy-naïve NSCLC patients.

Highlights

The most common form of lung cancer, non-small cell lung cancer (NSCLC), accounts for 85% of lung cancers and is a strong contributor of tumor morbidity and mortality worldwide (Bray et al 2018)
According to the fluorescence dot counts of epithelial and mesenchymal markers, hybrid E/M phenotype Circulating tumor cells (CTCs) were further divided into three subtypes, including E > m CTCs (Fig. 1D), e = m CTCs (Fig. 1E)and e < M CTCs (Fig. 1F)
A epithelial CTCs (E-CTC): epithelial marker +, mesenchymal marker, CD45, and DAPI + cells; B: hybrid E/mesenchymal CTCs (M-CTCs): epithelial marker +, mesenchymal marker +, CD45, and DAPI + cells; C M-CTCs: epithelial marker, mesenchymal marker +, CD45, and DAPI + cells; D E > m CTCs; E e = m CTCs; F e < M CTCs of hybrid E/M phenotype CTCs was 17 cells/5 mL, and the median number of E-CTCs and M-CTCs was 1 cells/5 mL

Summary

Introduction

The most common form of lung cancer, non-small cell lung cancer (NSCLC), accounts for 85% of lung cancers and is a strong contributor of tumor morbidity and mortality worldwide (Bray et al 2018). NSCLC patients may undergo surgery in early stages, while others may accept adjuvant chemotherapy and/or adjuvant radiotherapy before/after surgery. The rates of 5-year relapse and metastasis in early stage patients are about 20%. In the process of distant metastasis, CTCs undergo morphological transformation from proliferative phenotypes to migratory and invasive phenotypes, such as reversible epithelial mesenchymal (EMT) and mesenchymal epithelial transition (MET), termed as phenotypic plasticity(Tulchinsky et al 2019). EMT/MET has been reported in many studies and tumor types, including lung cancer (Lindsay et al 2017; Tayoun et al 2019). Further studies have reported that CTCs EMT/ MET phenotypes are related to patient prognosis. CTCs mesenchymal phenotype in breast cancer patients is associated with distant metastasis (Zhang et al 2017). More recent research showed that the CTCs epithelial phenotype with a restricted mesenchymal transition was strongly associated with lung metastases, while the CTC mesenchymal phenotype showed limited metastatic ability (Liu et al 2019)

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