Abstract 497: PIK3CA mutational status in circulating tumor cells (CTCs) and corresponding circulating tumor DNA in breast cancer patients
Authors: Tzanikou Elena , Markou Athina , Malamos Nikos , Georgoulias Vasilis , Lianidou Evi S. 
Published: July 2016

Abstract
AIMS: Molecular characterization of Circulating Tumor Cells (CTCs) and analysis of circulating tumor DNA (ctDNA) in cancer patients holds promise as an extremely powerful and reliable non-invasive clinical tool for the individual molecular profiling of each patient in real time. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is implicated in human cancers, and somatic mutations in the p110α catalytic subunit of PI3K, are very frequent and play a crucial role in response to molecular targeted therapies in breast cancer. In this study, we analyzed PIK3CA hotspot mutations (1633 G&gt;A, 3140 A&gt;G) in CTCs and corresponding ctDNA of early and metastatic breast cancer patients. We also examined whether there is a correlation between the presence of PIK3CA mutations in CTCs and ctDNA.
METHODS: We used our highly sensitive methodology for the detection of PIK3CA hotspot mutations in exons 9 (1633 G&gt;A) and 20 (3140 A&gt;G), based on a combination of allele-specific PCR, asymmetric rapid PCR and high resolution melting analysis (HRMA) (Markou et al, CCR 2014). We analyzed PIK3CA hotspot mutations in the EpCAM-positive CTCs fraction and the corresponding plasma samples of: i) a group of 19 patients with operable breast cancer, ii) a group of 40 breast cancer patients with verified metastasis and iii) 30 healthy female volunteers. All ctDNA samples (extracted from 2 ml plasma) were examined for their DNA quality; to verify DNA quality, primers specific for the wild type in exactly the same PIK3CA gene region for exon 9 were used to assess for hotspots mutations. The mutation status of PIK3CA gene in ctDNA samples was detected by the developed methodology exactly as previously described. All samples were analyzed in triplicate.
RESULTS: The assay is highly sensitive as it can detect 0.05% of mutated dsDNA in the presence of 99.95% wtDNA for both exons (9 and 20) and highly specific (0/30 healthy donors). PIK3CA hotspot mutations were identified in ctDNA in 12/40 (30%) of metastasis-verified breast cancer patients and 4/19 (21%) of operable breast cancer patients. In metastasis-verified breast cancer patients, the concordance between EpCAM- positive CTCs and ctDNA for 1633 G&gt;A mutation was 72.5%, whereas the corresponding concordance for the 3140 A&gt;G mutation was 97.5%. In operable breast cancer patients, the concordance between EpCAM-positive CTCs and ctDNA for the 1633 G&gt;A mutation was 80%, whereas the corresponding concordance for the 3140 A&gt;G mutation was 100%.
CONCLUSIONS: Detection of PIK3CA hotspot mutations in EpCAM-positive CTCs and corresponding ctDNA has shown a correlation both in metastasis-verified and operable breast cancer patients. We will further evaluate our findings in a large cohort of patients before and after treatment, to evaluate response to molecular targeted therapies in breast cancer.
Citation Format: Elena Tzanikou, Athina Markou, Nikos Malamos, Vasilis Georgoulias, Evi S. Lianidou. PIK3CA mutational status in circulating tumor cells (CTCs) and corresponding circulating tumor DNA in breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 497.