Integrative statistical analyses of multiple liquid biopsy analytes in metastatic breast cancer

Corinna Keup,Peter Hahn,Mitra Tewes,Markus Sprenger-Haussels,Vinay Suryaprakash,Oliver Hoffmann,Rainer Kimmig,Sabine Kasimir-Bauer,Hans-Christian Kolberg,Siegfried Hauch,Markus Storbeck

doi:10.1186/s13073-021-00902-1

Abstract

BackgroundSingle liquid biopsy analytes (LBAs) have been utilized for therapy selection in metastatic breast cancer (MBC). We performed integrative statistical analyses to examine the clinical relevance of using multiple LBAs: matched circulating tumor cell (CTC) mRNA, CTC genomic DNA (gDNA), extracellular vesicle (EV) mRNA, and cell-free DNA (cfDNA).MethodsBlood was drawn from 26 hormone receptor-positive, HER2-negative MBC patients. CTC mRNA and EV mRNA were analyzed using a multi-marker qPCR. Plasma from CTC-depleted blood was utilized for cfDNA isolation. gDNA from CTCs was isolated from mRNA-depleted CTC lysates. CTC gDNA and cfDNA were analyzed by targeted sequencing. Hierarchical clustering was performed within each analyte, and its results were combined into a score termed Evaluation of multiple Liquid biopsy analytes In Metastatic breast cancer patients All from one blood sample (ELIMA.score), which calculates the contribution of each analyte to the overall survival prediction. Singular value decomposition (SVD), mutual information calculation, k-means clustering, and graph-theoretic analysis were conducted to elucidate the dependence between individual analytes.ResultsA combination of two/three/four LBAs increased the prevalence of patients with actionable signals. Aggregating the results of hierarchical clustering of individual LBAs into the ELIMA.score resulted in a highly significant correlation with overall survival, thereby bolstering evidence for the additive value of using multiple LBAs. Computation of mutual information indicated that none of the LBAs is independent of the others, but the ability of a single LBA to describe the others is rather limited—only CTC gDNA could partially describe the other three LBAs. SVD revealed that the strongest singular vectors originate from all four LBAs, but a majority originated from CTC gDNA. After k-means clustering of patients based on parameters of all four LBAs, the graph-theoretic analysis revealed CTC ERBB2 variants only in patients belonging to one particular cluster.ConclusionsThe additional benefits of using all four LBAs were objectively demonstrated in this pilot study, which also indicated a relative dominance of CTC gDNA over the other LBAs. Consequently, a multi-parametric liquid biopsy approach deconvolutes the genomic and transcriptomic complexity and should be considered in clinical practice.

Highlights

Single liquid biopsy analytes (LBAs) have been utilized for therapy selection in metastatic breast cancer (MBC)
The case study of a HR+ MBC patient with serial liquid biopsies across treatment over 4 years showed the correlation of single circulating tumor cells (CTCs) and cellfree DNA (cfDNA) copy number variants and mutations, but the authors argued that only the analysis of variants in single CTCs deconvolutes the subclonal evolution in cellular resolution [52]
The surplus information, obtained from the multiparametric liquid biopsy approach by integrating the four LBAs, was mirrored by the prognostic value of the ELIMA.score and the improved sensitivity for actionable markers, and by the low mutual information values and a fairly even spread of data points across the dimensions when k-means clustering based on all four LBAs was carried out. k-means clustering and graph-theoretical analysis of the resulting clusters further highlighted the differences and identified some unique features across the different LBAs in the four clusters, thereby accentuating the relevance of the integration of all four LBAs

Summary

Introduction

Single liquid biopsy analytes (LBAs) have been utilized for therapy selection in metastatic breast cancer (MBC). We performed integrative statistical analyses to examine the clinical relevance of using multiple LBAs: matched circulating tumor cell (CTC) mRNA, CTC genomic DNA (gDNA), extracellular vesicle (EV) mRNA, and cellfree DNA (cfDNA). In breast cancer (BC), the leading form of cancer in women worldwide [2], diverse liquid biopsy analytes (LBAs) have been proven to harbor relevance in clinical practice. For example: The number of circulating tumor cells (CTCs) [3, 4] as well as the concentration of cell-free tumor DNA (ctDNA) [5] has been proven to correlate significantly with overall survival (OS). Profiling CTCs revealed HER2 overexpression in the blood of patients with HER2-negative primary tumors, and clinical effectiveness has not yet been concretely proven, it points towards a possible targeted antiHER2 therapy based on blood testing [7,8,9].

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