The dynamic range of circulating tumor DNA in metastatic breast cancer

Abstract

Introduction: The management of metastatic breast cancer needs improvement. As clinical evaluation is not very accurate in determining the progression of disease, the analysis of circulating tumor DNA (ctDNA) has evolved to a promising noninvasive marker of disease evolution. Indeed, ctDNA was reported to represent a highly sensitive biomarker of metastatic cancer disease directly reflecting tumor burden and dynamics. However, at present little is known about the dynamic range of ctDNA in patients with metastatic breast cancer.

Methods: In this study, 74 plasma DNA samples from 58 patients with metastasized breast cancer were analyzed with a microfluidic device to determine the plasma DNA size distribution and copy number changes in the plasma were identified by whole-genome sequencing (plasma-Seq). Furthermore, in an index patient we conducted whole-genome, exome, or targeted deep sequencing of the primary tumor, metastases, and circulating tumor cells (CTCs). Deep sequencing was done to accurately determine the allele fraction (AFs) of mutated DNA fragments.

Results: Although all patients had metastatic disease, plasma analyses demonstrated highly variable AFs of mutant fragments. We analyzed an index patient with more than 100,000 CTCs in detail. We first conducted whole-genome, exome, or targeted deep sequencing of four different regions from the primary tumor and three metastatic lymph node regions, which enabled us to establish the phylogenetic relationships of these lesions, which were consistent with a genetically homogeneous cancer. Subsequent analyses of 551 CTCs confirmed the genetically homogeneous cancer in three serial blood analyses. However, the AFs of ctDNA were only 2% to 3% in each analysis, neither reflecting the tumor burden nor the dynamics of this progressive disease. These results together with high-resolution plasma DNA fragment sizing suggested that differences in phagocytosis and DNA degradation mechanisms likely explain the variable occurrence of mutated DNA fragments in the blood of patients with cancer.

Conclusions: The dynamic range of ctDNA varies substantially in patients with metastatic breast cancer. This has important implications for the use of ctDNA as a predictive and prognostic biomarker.

Figure 1

Timeline of the clinical course of the index patient and results obtained from the analyzed primary tumor and metastatic lesions. (a) The timeline starts with a sketch illustrating the localization of the lesions we subjected to our analyses, that is primary tumor lesions A, B, C, and D and lymph node metastases LN15, LN17, and LNA from the right axilla as observed at the time of diagnosis. In the timeline, dates when clinical progress was noted are indicated with a blue bar and the time points of blood collections (B) by a red bar (for details see text). (b) Copy number profiles obtained by whole-genome sequencing from tumor C and lymph node metastasis LN17. The X-axis shows the chromosome, the Y-axis indicates log₂-ratios. (c) Mutation frequencies of genes PCDH20, OR4X1, ALK, DNPEP, SH3TC2, DDR2, MLL3, and PIK3CA as established by targeted deep sequencing in the tumor lesions and lymph node metastases. (d) The clonal evolutionary relationships of the analyzed lesions based on the frequency of single nucleotide variants (SNVs) as shown in (c). The arrow indicates an increase in the frequency of the respective SNV, the `+' a newly occurred SNV. The three lymph node metastases are indicated together in the circle labeled `LN'.

Figure 2

Analyses of circulating tumor cells (CTCs) from the index patient. (a) Whole-genome sequencing profile from a single CTC. (b) Left panel: heat maps from the copy number changes (red: overrepresentation, blue: underrepresentation) demonstrating the similarities between the various analyzed samples. Right panel: hierarchical clustering (Manhattan distance, complete agglomeration), the dendrogram illustrates the clonal relationships.

Figure 3

Plasma DNA analyses from the index patient. (a) Sizing of the plasma DNA fragments with a microfluidic device, that is the Agilent 2100 Bioanalyzer. All three plasma samples from the index patient (P1, P2, and P3, which correspond to the first, second, and third blood sample) demonstrated enrichment of short DNA fragments (size range 85 to 250 bps), whereas longer DNA size fragments (250 to 450 bps) were barely present. (b) Whole-genome sequencing (plasma-Seq) profiles of the three blood samples.

Figure 4

Exemplary plasma DNA analyses from patients B49 and B60 and plasma DNA copy number patterns from a panel of patients with metastatic breast cancer. (a) Patient B60 demonstrated an enrichment of plasma DNA fragments at 160 bps whereas B49 had plasma DNA fragments at about 315 bps in addition. (b) Corresponding plasma-Seq profiles of B60 and B49. (c) Copy number patterns established from a panel of patients with metastatic breast cancer. Depicted are recurrent aberrations (upper panel gains, lower panel losses), the Y-axis represents relative abundance of respective aberrations of unbalanced copy number profiles. Data from patients with balanced copy number profiles were not included. These analyses revealed losses of 1p, 8p, 13q, and 16q, and gains of 1q, 8q, 16p, and 17q.