Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling

Giuseppa De Luca,Anna Garuti,Manlio Ferrarini,Franca Carli,George A Calin,Barbara Cardinali,Simona Zupo,Carlotta Mazzitelli,Sonia Lastraioli,Mariella Dono,Lucia Del Mastro

doi:10.3390/ijms21124364

Abstract

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2–5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.

Highlights

MDA-MB-231 (MDA) and MCF-7 cell lines were employed as surrogate Breast Cancer (BC) Circulating Tumor Cells (CTCs) and used in spiking-in experiments by addition to a healthy donor blood sample before DEPArrayTM isolation
We found that 7/13 BC CTCs samples had a mutation occurring; mainly in the TP53 and PIK3CA genes, as these genes are the most frequently mutated in BC [37,38,39]
This study has demonstrated, for the first time, the potential application of a molecular tagging Next-Generation Sequencing (NGS) panel, which is already standardized for other liquid biopsy purposes, as a feasible mutational approach for CTCs sequencing

Summary

Introduction

Circulating Tumor Cells (CTCs) in the bloodstream, which originate from primary and/or metastatic tumors, are both phenotypically and molecularly highly heterogeneous [1,2].An increasing number of data have confirmed the role of CTCs count and their molecular characterization in patient prognosis, relapse prediction, and therapy outcome, indicating that CTCs represent an attractive blood-based biomarker with high potential for understanding the clinical behavior of the associated disease [3,4,5,6,7,8,9,10].To date, major efforts towards the molecular characterization of CTCs have been focused on the optimization of genetic analysis protocols, mainly including Whole Genome Amplification (WGA) as a first step, which allows for the isolation of an acceptable amount of DNA from few starting molecules [11,12,13,14,15,16]. Different studies have demonstrated a series of drawbacks arising from WGA procedures, such as allelic dropout, chimeric molecules, low uniformity, and polymerase errors, which lead to bias in the interpretation of molecular data [17,18,19,20,21]. These biases are partly caused by an unavoidable delay in the mutational profile analysis of CTCs in routine laboratory practice. Different workflows have been developed to provide alternative options to the traditional techniques which do not require this preamplification step, instead of utilizing downstream whole-genome or targeted sequencing [22,23,24]

Methods

Results

Discussion

Conclusion