Divergence between high metastatic tumor burden and low circulating tumor DNA concentration in metastasized breast cancer. M. Heidary1, M. Auer1, P. Ulz1, E. Heitzer1, E. Petru2, C. Gasch3, S. Riethdorf3, O. Mauermann3, I. Lafer1, G. Pristauz2, S. Lax4, K. Pantel3, JB. Geigl1, MR. Speicher1 1) Institute of Human Genetics, Medical University of Graz, Harrachgasse 21/8, A-8010 Graz, Austria; 2) Department of Obstetrics and Gynecology, Medical University of Graz, Auenbruggerplatz 14, A-8036 Graz, Austria; 3) Institute of Tumor Biology, University Medical Center Hamburg Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany; 4) Department of Pathology, General Hospital Graz West, Goestingerstrasse 22, A-8020 Graz, Austria.

   Recently there has been considerable interest in circulating tumor DNA (ctDNA) as non-invasive biomarker in patients with cancer. Although recent studies have shown that ctDNA is an informative and highly sensitive biomarker for monitoring the tumor burden dynamics and treatment responses in metastatic cancer patients, current knowledge of the dynamic range of ctDNA in patients with metastatic breast cancer is limited. To address this issue, we studied the role of ctDNA in patients with metastatic breast cancer and analyzed 74 plasma DNA samples from 58 patients. We used a microfluidic device to analyze size distribution of the plasma DNA and whole genome sequencing (plasma-Seq) to identify copy number changes in the plasma. Highly variable AFs of mutant fragments were detected through analyses of 74 plasma samples from 58 patients, and did not reflect the tumor burden in all cases. To show that tumor burden is not necessarily reflected by mutated AFs we analyzed an index patient in detail. This index patient had more than 100,000 circulating tumor cells (CTCs) in three serial blood analyses and we comprehensively analyzed the primary tumor, metastatic deposits, single and pools of CTCs, and ctDNA using whole-genome, exome, or targeted deep sequencing. Accurate evaluation of the allele fraction (AFs) of mutated DNA fragments was performed using targeted deep-sequencing. Sequencing of four different regions of the primary tumor and three metastatic lymph node regions revealed genetically homogeneous cancer disease. Subsequently, detailed analyses of 551 CTCs demonstrated a high degree of similarities to the primary tumor and metastases and verified the genetically homogeneous cancer. However, despite the extraordinarily high CTC number ctDNA analyses detected a very low mutant AF of only 2-3% in each of the serial samples, which did not reflect the tumor burden or the dynamics of this progressive disease. These data suggest a highly variable range of ctDNA in patients with metastatic breast cancer, which may have an impact on the use of ctDNA as a predictive and prognostic biomarker.

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