A fully automated assay to detect the expression of pan-cytokeratins and of EML4-ALK fusion protein in circulating tumour cells (CTCs) predicts outcome of non-small cell lung cancer (NSCLC) patients

Elisabetta Rossi; Michele Aieta; Alfredo Tartarone; Aldo Pezzuto; Antonella Facchinetti; Daniele Santini; Paola Ulivi; Vienna Ludovini; Luciana Possidente; Pasquale Fiduccia; Nadia Minicuci; Rita Zamarchi

doi:10.21037/tlcr-20-855

A fully automated assay to detect the expression of pan-cytokeratins and of EML4-ALK fusion protein in circulating tumour cells (CTCs) predicts outcome of non-small cell lung cancer (NSCLC) patients

Transl Lung Cancer Res. 2021 Jan;10(1):80-92. doi: 10.21037/tlcr-20-855.

Authors

Affiliations

¹ Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy.
² Veneto Institute of Oncology IOV - IRCCS, Padua, Italy.
³ CROB-IRCCS - Rionero in Vulture (PZ), Italy.
⁴ Department of Cardiopulmonary and Vascular Science, S. Andrea Hospital-Sapienza University, Roma, Italy.
⁵ Medical Oncology Unit, Campus Bio-Medico University, Rome, Italy.
⁶ Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola (FC), Italy.
⁷ Department of Medical Oncology, Santa Maria della Misericordia Hospital, Perugia, Italy.
⁸ CNR, Neuroscience Institute, Padua, Italy.

Abstract

Background: In advanced non-small cell lung cancer (NSCLC) a recent meta-analysis confirms circulating tumour cells (CTCs) as an independent prognostic indicator of progression-free survival (PFS) and overall survival (OS). However, further investigations are necessary to predict and dynamically monitor the therapy in NSCLC patients using CTCs. To this aim, we combined the classical standard assay (SA) with an expanded cytokeratins profile (EA) and quantified the expression of EML4-ALK fusion protein in CTCs.

Methods: The CellSearch (CS) platform-first marked in vitro diagnostic use (IVD) from Food and Drug Administration (FDA), and "gold standard" for quantifying CTCs - detects EpCAM and cytokeratins (CKs) 8, 18, and 19. Since NSCLC shows different CKs profile, we customized the SA, to recognize CK 4, 5, 6, 7, 8, 10, 13, 14, 18, and 19 (EA). Using both assays we designed a prospective, multi-center study, primarily aimed to enumerate CTCs in advanced NSCLC. Secondarily, we developed an integration of the EA to quantify the expression of EML4-ALK fusion protein in CTCs, and correlated them with PFS and OS.

Results: EA identified a significantly much more number of CTC-positive patients (115 out of 180) than SA (103 out of 192; Chi-square =4.0179, with 1 degrees of freedom, P=0.04502). Similar to SA, EA levels were still associated with patient' outcomes. Furthermore, the expression of EML4-ALK on CTCs allowed stratifying NSCLC patients according to a statistically significant difference in PFS.

Conclusions: We proposed here two novel automated tests, to characterize the expression of specific molecules on CTCs. We demonstrated that these integrated assays are robust and actionable in prospective clinical studies, and in the future could allow clinicians to improve both choice and length of treatment in individual NSCLC patient.

Keywords: EML4-ALK; Non-small cell lung cancer (NSCLC); circulating tumour cells (CTCs); cytokeratins (CK); liquid biopsy.