Emmanuel Acheampong1,2, Michael Morici1,2, Afaf Abed1,2,3, Samantha Bowyer4,5,3, Du-Bois Asante1,2, Weitao Lin1,2,6, Michael Millward1,2,5,3, Elin S Gray7,8, Aaron B Beasley1,2. 1. School of Medical and Health Sciences, Edith Cowan University, Perth, Joondalup, WA, 6027, Australia. 2. Centre for Precision Health, Edith Cowan University, Joondalup, WA, 6027, Australia. 3. Linear Clinical Research, Hospital Avenue, Nedlands, WA, 6009, Australia. 4. Department of Medical Oncology, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, WA, 6009, Australia. 5. School of Medicine and Pharmacology, University of Western Australia, Crawley, WA, 6009, Australia. 6. Harry Perkins Institute of Medical Research, Nedlands, WA, 6009, Australia. 7. School of Medical and Health Sciences, Edith Cowan University, Perth, Joondalup, WA, 6027, Australia. e.gray@ecu.edu.au. 8. Centre for Precision Health, Edith Cowan University, Joondalup, WA, 6027, Australia. e.gray@ecu.edu.au.
Abstract
BACKGROUND: Circulating tumour cells (CTCs) are attractive "liquid biopsy" candidates that could provide insights into the different phenotypes of tumours present within a patient. The epithelial-to-mesenchymal transition (EMT) of CTCs is considered a critical step in tumour metastasis; however, it may confound traditional epithelial feature-based CTC isolation and detection. We applied single-cell copy number alteration (CNA) analysis for the identification of genomic alterations to confirm the neoplastic nature of circulating cells with only mesenchymal phenotypes. METHODS: We isolated CTCs from blood samples collected from 46 NSCLC patients using the Parsortix system. Enriched cells were subjected to immunofluorescent staining for CTC identification using a multi-marker panel comprising both epithelial and mesenchymal markers. A subset of isolated CTCs was subjected to whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for the analysis of copy number alterations (CNAs). RESULTS: CTCs were detected in 16/46 (34.8%) patients, inclusive of CK+/EpCAM+ CTCs (3/46, 6.5%) and Vim+ CTCs (13/46, 28.3%). Clusters of Vim+ cells were detected in 8 samples, which constitutes 50% of the total number of NSCLC patients with CTCs. No patients had detectable hybrid CK+/EpCAM+/Vim+ cells. All of the tested CK+/EpCAM+ CTCs and 7/8 Vim+ CTCs or CTC clusters carried CNAs confirming their neoplastic nature. Notably, the Vim+ cluster with no CNAs was characterised by spindle morphology and, therefore, defined as normal mesenchymal circulating cells. CONCLUSION: Our results revealed that CK-negative, vimentin-expressing cells represent a large proportion of CTCs detected in NSCLC patients, which are likely missed by standard epithelial-marker-dependent CTC categorisation.
BACKGROUND: Circulating tumour cells (CTCs) are attractive "liquid biopsy" candidates that could provide insights into the different phenotypes of tumours present within a patient. The epithelial-to-mesenchymal transition (EMT) of CTCs is considered a critical step in tumour metastasis; however, it may confound traditional epithelial feature-based CTC isolation and detection. We applied single-cell copy number alteration (CNA) analysis for the identification of genomic alterations to confirm the neoplastic nature of circulating cells with only mesenchymal phenotypes. METHODS: We isolated CTCs from blood samples collected from 46 NSCLC patients using the Parsortix system. Enriched cells were subjected to immunofluorescent staining for CTC identification using a multi-marker panel comprising both epithelial and mesenchymal markers. A subset of isolated CTCs was subjected to whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for the analysis of copy number alterations (CNAs). RESULTS: CTCs were detected in 16/46 (34.8%) patients, inclusive of CK+/EpCAM+ CTCs (3/46, 6.5%) and Vim+ CTCs (13/46, 28.3%). Clusters of Vim+ cells were detected in 8 samples, which constitutes 50% of the total number of NSCLC patients with CTCs. No patients had detectable hybrid CK+/EpCAM+/Vim+ cells. All of the tested CK+/EpCAM+ CTCs and 7/8 Vim+ CTCs or CTC clusters carried CNAs confirming their neoplastic nature. Notably, the Vim+ cluster with no CNAs was characterised by spindle morphology and, therefore, defined as normal mesenchymal circulating cells. CONCLUSION: Our results revealed that CK-negative, vimentin-expressing cells represent a large proportion of CTCs detected in NSCLC patients, which are likely missed by standard epithelial-marker-dependent CTC categorisation.
Authors: F-C Bidard; C Mathiot; A Degeorges; M-C Etienne-Grimaldi; R Delva; X Pivot; C Veyret; L Bergougnoux; P de Cremoux; G Milano; J-Y Pierga Journal: Ann Oncol Date: 2010-03-16 Impact factor: 32.976
Authors: Hossein Borghaei; Luis Paz-Ares; Leora Horn; David R Spigel; Martin Steins; Neal E Ready; Laura Q Chow; Everett E Vokes; Enriqueta Felip; Esther Holgado; Fabrice Barlesi; Martin Kohlhäufl; Oscar Arrieta; Marco Angelo Burgio; Jérôme Fayette; Hervé Lena; Elena Poddubskaya; David E Gerber; Scott N Gettinger; Charles M Rudin; Naiyer Rizvi; Lucio Crinò; George R Blumenschein; Scott J Antonia; Cécile Dorange; Christopher T Harbison; Friedrich Graf Finckenstein; Julie R Brahmer Journal: N Engl J Med Date: 2015-09-27 Impact factor: 91.245
Authors: Emmanuel Acheampong; Afaf Abed; Michael Morici; Isaacs Spencer; Aaron B Beasley; Samantha Bowyer; Du-Bois Asante; Chris Lomma; Weitao Lin; Michael Millward; Elin S Gray Journal: Transl Lung Cancer Res Date: 2022-03