Development and Validation of a Multi-marker Liquid Bead Array Assay for the Simultaneous Detection of and Hotspot Mutations in Single Circulating Tumor Cells (CTCs)

Overview

Journal Heliyon

Specialty Social Sciences

Date 2024 Oct 10

PMID 39386783

Authors

Dimitra Stergiopoulou

Vassilis Georgoulias

Athina Markou

Evi Lianidou

Affiliations

Soon will be listed here.

Abstract

Background: and mutations are associated with progression and therapy resistance in metastatic breast cancer (MBC). CTCs are highly heterogeneous and their analysis at single cell level can provide unique information for mutational profiling and the existence of different sub-clones related to tumor progression. We have developed a novel multi-marker liquid bead array assay based on combination of an enzymatic mutation enrichment method, multiplex PCR-based assay, and liquid bead array technology for the simultaneous detection of and hotspot mutations in liquid biopsy samples. We focus on single CTCs, however the assay can be used for bulk CTC and ctDNA analysis.

Materials And Methods: Single CTCs were isolated from an ER+/HER2+ MBC patient from CellSearch® cartridges using the VyCAP Puncher System and subjected to whole genome amplification followed by nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO) enrichment. The assay was validated for analytical sensitivity and specificity for the simultaneous detection of () and (Y537S) mutations in single CTCs, while its clinical performance was evaluated on 22 single CTCs and three single white blood cells (WBCs).

Results: The developed multi-marker liquid bead array assay is novel, highly specific and sensitive for both mutation panels. The assay can reliably detect mutation-allelic-frequencies (MAFs) as low as 0.1 %. The presence of and mutations was detected in 13.6 % and 72.7 % of single CTCs, respectively. The developed assay is sample-saving since it requires only 2 μL of amplified DNA to check for nine hotspot and mutations in a single cell. The developed liquid bead array assay (Luminex, US), based on a 96 microwell plate format, enables the simultaneous analysis of 96 single cells.

Conclusions: The developed novel multi-marker liquid bead array assay for the simultaneous detection of and hotspot mutations in single CTCs is highly specific, highly sensitive, high-throughput, and sample-, cost-, and time-saving. This multi-marker liquid bead array assay can be extended to detect up to 100 mutations in many genes at once and can be applied for bulk CTC and ctDNA analysis.

References

Lampignano R, Yang L, Neumann M, Franken A, Fehm T, Niederacher D . A Novel Workflow to Enrich and Isolate Patient-Matched EpCAM and EpCAM CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer. Int J Mol Sci. 2017; 18(9). PMC: 5618534. DOI: 10.3390/ijms18091885. View

Deng G, Krishnakumar S, Powell A, Zhang H, Mindrinos M, Telli M . Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow. BMC Cancer. 2014; 14:456. PMC: 4071027. DOI: 10.1186/1471-2407-14-456. View

Casado-Pelaez M, Bueno-Costa A, Esteller M . Single cell cancer epigenetics. Trends Cancer. 2022; 8(10):820-838. DOI: 10.1016/j.trecan.2022.06.005. View

Parisi C, Markou A, Strati A, Kasimir-Bauer S, Lianidou E . Development and Validation of Multiplex Liquid Bead Array Assay for the Simultaneous Expression of 14 Genes in Circulating Tumor Cells. Anal Chem. 2019; 91(5):3443-3451. DOI: 10.1021/acs.analchem.8b04975. View

Stathopoulou A, Ntoulia M, Perraki M, Apostolaki S, Mavroudis D, Malamos N . A highly specific real-time RT-PCR method for the quantitative determination of CK-19 mRNA positive cells in peripheral blood of patients with operable breast cancer. Int J Cancer. 2006; 119(7):1654-9. DOI: 10.1002/ijc.22017. View

Alexovic M, Ulicna C, Sabo J, Davalieva K . Human peripheral blood mononuclear cells as a valuable source of disease-related biomarkers: Evidence from comparative proteomics studies. Proteomics Clin Appl. 2023; 18(2):e2300072. DOI: 10.1002/prca.202300072. View

Markou A, Farkona S, Schiza C, Efstathiou T, Kounelis S, Malamos N . PIK3CA mutational status in circulating tumor cells can change during disease recurrence or progression in patients with breast cancer. Clin Cancer Res. 2014; 20(22):5823-34. DOI: 10.1158/1078-0432.CCR-14-0149. View

Fusco N, Malapelle U, Fassan M, Marchio C, Buglioni S, Zupo S . Mutations as a Molecular Target for Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer. Front Oncol. 2021; 11:644737. PMC: 8027489. DOI: 10.3389/fonc.2021.644737. View

Cristofanilli M, Pierga J, Reuben J, Rademaker A, Davis A, Peeters D . The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper. Crit Rev Oncol Hematol. 2019; 134:39-45. DOI: 10.1016/j.critrevonc.2018.12.004. View

10.

Ignatiadis M, Sledge G, Jeffrey S . Liquid biopsy enters the clinic - implementation issues and future challenges. Nat Rev Clin Oncol. 2021; 18(5):297-312. DOI: 10.1038/s41571-020-00457-x. View

11.

Graham H, Chandler D, Dunbar S . The genesis and evolution of bead-based multiplexing. Methods. 2019; 158:2-11. DOI: 10.1016/j.ymeth.2019.01.007. View

12.

Brett J, Spring L, Bardia A, Wander S . ESR1 mutation as an emerging clinical biomarker in metastatic hormone receptor-positive breast cancer. Breast Cancer Res. 2021; 23(1):85. PMC: 8365900. DOI: 10.1186/s13058-021-01462-3. View

13.

Shaw J, Guttery D, Hills A, Fernandez-Garcia D, Page K, Rosales B . Mutation Analysis of Cell-Free DNA and Single Circulating Tumor Cells in Metastatic Breast Cancer Patients with High Circulating Tumor Cell Counts. Clin Cancer Res. 2016; 23(1):88-96. PMC: 6241844. DOI: 10.1158/1078-0432.CCR-16-0825. View

14.

Darbeheshti F, Yu F, Makrigiorgos G . Pre-PCR Mutation-Enrichment Methods for Liquid Biopsy Applications. Cancers (Basel). 2022; 14(13). PMC: 9264780. DOI: 10.3390/cancers14133143. View

15.

Dawson S . Characterizing the Cancer Genome in Blood. Cold Spring Harb Perspect Med. 2018; 9(4). PMC: 6444693. DOI: 10.1101/cshperspect.a026880. View

16.

De Mattos-Arruda L . mutation inhibition in hormone receptor-positive breast cancer: time has come. ESMO Open. 2020; 5(4). PMC: 7437706. DOI: 10.1136/esmoopen-2020-000890. View

17.

Ivanova E, Ward A, Wiegmans A, Richard D . Circulating Tumor Cells in Metastatic Breast Cancer: From Genome Instability to Metastasis. Front Mol Biosci. 2020; 7:134. PMC: 7378584. DOI: 10.3389/fmolb.2020.00134. View

18.

Stergiopoulou D, Markou A, Strati A, Zavridou M, Tzanikou E, Mastoraki S . Comprehensive liquid biopsy analysis as a tool for the early detection of minimal residual disease in breast cancer. Sci Rep. 2023; 13(1):1258. PMC: 9870904. DOI: 10.1038/s41598-022-25400-1. View

19.

Bidard F, Hardy-Bessard A, Dalenc F, Bachelot T, Pierga J, de La Motte Rouge T . Switch to fulvestrant and palbociclib versus no switch in advanced breast cancer with rising ESR1 mutation during aromatase inhibitor and palbociclib therapy (PADA-1): a randomised, open-label, multicentre, phase 3 trial. Lancet Oncol. 2022; 23(11):1367-1377. DOI: 10.1016/S1470-2045(22)00555-1. View

20.

Radfar P, Aboulkheyr Es H, Salomon R, Kulasinghe A, Ramalingam N, Sarafraz-Yazdi E . Single-cell analysis of circulating tumour cells: enabling technologies and clinical applications. Trends Biotechnol. 2022; 40(9):1041-1060. DOI: 10.1016/j.tibtech.2022.02.004. View