Abstract PD3-8: Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients
Authors: Bidard Francois-Clement , Madic Jordan , Kiialainen Anna , Birzele Fabian , Ramey Guillemette , Leroy Quentin , Rio Frio Thomas , Raynal Virginie , Bernard Virginie , Lermine Alban , Clausen Inga , Giroud Nicolas , Schmucki Roland , Horn Carsten , Spleiss Olivia , Lantz Olivier , Stern Marc-Henri , Weisser Martin , Lebofsky Ronald , Pierga Jean-Yves 
Published: May 2015

Abstract
Background
Preliminary reports suggested that circulating tumor DNA (ctDNA) can be used as a prognostic marker in a way akin to circulating tumor cells (CTC) in metastatic breast cancer patients. However ctDNA detection is often performed on multiple mutations, combining heterogeneous techniques. Here we used the high prevalence of TP53 mutations in triple-negative metastatic breast cancer (TNMBC) to compare CTC and ctDNA detection rates and prognostic value.
Methods
A cohort of 40 patients treated at the Institut Curie (Paris, France) was enrolled before starting a new line of treatment for TNMBC. CTC were detected by the CellSearch system (in 7.5 mL of blood). Using massively parallel sequencing (NGS), TP53 mutations were first characterized in tumor tissue, then in plasma DNA extracted from fresh frozen plasma samples (from 15-20 mL of blood). ctDNA detection was performed using high depth targeted sequencing using two platforms in parallel (Illumina HiSeq 2500 and Roche 454). Libraries for Illumina were prepared following the TAm-Seq procedure (Forshew et al, Sci Transl Med 2012), with preamplification of all coding TP53 exons and flanking untranslated regions followed by both paired-end 150bp Illumina and 454/Roche sequencing. CTC, ctDNA and usual patient characteristics were correlated with time to progression (TTP) and overall survival (OS).
Results
Archived tumor (FFPE or frozen) tissue was available for 36 patients, and 31 were successfully sequenced: TP53 mutations were found in 27 patients. As measured on the Illumina platform, ctDNA was detected in 21/27 patients (81%), ranging from 48 to 648,000 copies/mL of plasma (median 1620). Mutant allele fraction in circulating cell-free DNA ranged from 2 to 70% (median 5%). Comparison between ctDNA levels measured by Illumina and 454/Roche platforms in plasma displayed a good correlation (R2 = 0.903), with a single discordance. ≥1 CTC were detected in 19 of these 27 patients (70%). Strikingly, high ctDNA levels had prognostic impact neither on OS, nor on TTP, whatever the dataset used (Illumina or 454) whereas CTC≥5/7.5 mL were correlated with OS (p=0.04) and marginally with TTP (p=0.06). Other known usual factors, such as poor performance status, elevated LDH and number of previous treatment lines had also significant prognostic factors in this cohort. CTC and ctDNA early changes during treatment were available for 12 patients and changes (increase/decrease) of the two biomarkers were globally similar.
Conclusion
Demonstrating a good sensitivity (81%), ctDNA by the TAm-Seq is more frequently detected than CTCs in the 27 TNMBC with TP53 mutations. The observed correlation between the 2 massively parallel sequencing approaches suggested that ctDNA levels data were quantitative. In contrast to other usual prognostic factors, baseline ctDNA level did not demonstrate a prognostic impact,in this proof-of-principle study, suggesting that mechanisms of ctDNA release in TNMBC rely on biological features that do not dramatically impact patient’s outcome.
Citation Format: Francois-Clement Bidard, Jordan Madic, Anna Kiialainen, Fabian Birzele, Guillemette Ramey, Quentin Leroy, Thomas Rio Frio, Virginie Raynal, Virginie Bernard, Alban Lermine, Inga Clausen, Nicolas Giroud, Roland Schmucki, Carsten Horn, Olivia Spleiss, Olivier Lantz, Marc-Henri Stern, Martin Weisser, Ronald Lebofsky, Jean-Yves Pierga. Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-8.