Purpose: Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while PIK3CA somatic mutations play a crucial role in therapy response. We investigated the presence of PIK3CA mutations in CTC and whether this is associated with clinical outcome.Experimental Design: We developed and validated an ultrasensitive methodology for the detection of PIK3CA mutations that is based on a combination of allele-specific, asymmetric rapid PCR and melting analysis. We analyzed PIK3CA hotspot mutations in: (i) a training group consisting of EpCAM-positive CTC fraction from 37 patients with clinically confirmed metastasis, and 26 healthy female volunteers and 15 primary breast tumor tissues and (ii) an independent group consisting of EpCAM-positive CTC fraction from 57 metastatic and 118 operable breast cancer patients and 76 corresponding primary tumors.Results: The assay could detect 0.05% of mutated dsDNA in the presence of 99.95% wtDNA for both exons (9 and 20) and was highly specific (0/26 healthy donors). PIK3CA mutations were identified in EpCAM-positive CTC in 20 of 57(35.1%) and in 23 of 118 (19.5%) patients with metastatic and operable breast cancer, and in 45 of 76(59.2%) corresponding FFPEs. Our data indicate that PIK3CA mutational status in CTCs can change during disease progression and is associated with worse survival (P ¼ 0.047).Conclusions: PIK3CA hotspot mutations are present at a relatively high frequency in CTCs and their presence is associated with worse survival in patients with breast cancer with metastasis. Evaluation of PIK3CA mutational status in CTCs is a strategy with potential clinical application.

Introduction: Molecular characterization of CTC is crucial for the investigation of molecular targeted therapies. PIK3CA somatic mutations play a crucial role in response to molecular targeted therapies. We detected PIK3CA mutations at a high frequency in CTC after developing and validating an ultra-sensitive methodology, based on a combination of allele-specific, asymmetric rapid PCR and melting analysis. Patient and Methods: After optimizing and validating our assay in terms of sensitivity, specificity and robustness, we detected PIK3CA hotspot mutations in EpCAM positive CTCs from 57 metastatic and 118 early breast cancer patients, 26 healthy individuals and 76 corresponding primary tumors. Rapid Real-PCR and melting were performed in triplicate for all samples in the LightScanner32 (Idaho USA), in the presence of LC-Green Plus saturating dye. Results: The assay could detect 0.05% of mutated dsDNA in the presence of 99.95% wtDNA for both exons and was highly specific (0/26 healthy). We identified PIK3CA mutations in EpCAM positive CTC in 20/57(35.1%) metastatic and in 23/118(19.5%) operable breast cancer patients. In corresponding primary tumors, 45/76(59.2%) samples were positive. Patients with verified metastasis carrying PIK3CA mutations on CTC had significant shorter OS than those without. Conclusions: We report for the first time that PIK3CA hotspot mutations are present at a relatively high frequency in CTC both in metastatic and operable breast cancer. The presence of PIK3CA mutations in CTC is associated with worse survival in patients with verified metastasis. Evaluation of PIK3CA mutational status on CTCs is a strategy with potential clinical application. Citation Format: Athina N. Markou, Sofia Farkona, Christina Schiza, Antonia Eftathiou, Nikolaos Malamos, Vassilis Georgoulias, Evi Lianidou. PIK3CA hotspot mutations are present at a relatively high frequency in CTCs of operable and metastatic breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3067. doi:10.1158/1538-7445.AM2014-3067

Introduction: Somatic mutations in the gene encoding for the phosphatidyl-inositol 3-kinase (PI3K) catalytic subunit, PIK3CA, have been discovered in many different human cancers. We have recently developed a high-resolution melting curve analysis method for the detection of the three hotspot mutations of PIK3CA gene (Vorkas et al, J of Mol Diagn 2010). The aim of our study was to evaluate this method for the presence of PIK3CA mutations in cell-free DNA (cfDNA) circulating in plasma of breast cancer patients and healthy individuals. Patients and methods: cfDNA was isolated from plasma (200μL) of 30 patients with operable breast cancer, 45 patients with metastatic breast cancer and 12 healthy donors. After DNA extraction, real-time PCR was performed in triplicate for all samples in the LightCycler (Roche), in the presence of LC-Green Plus saturating dye (Idaho Technology Inc.). High-resolution melting curves were obtained with HR-1 High Resolution Melter (Idaho Technology Inc.). Results: We identified 15 cfDNA samples mutated out of 75 tested (20%). 3/75 (4%) cfDNA samples were mutated within the exon 9 amplified region, and 12/75 (16%) cfDNA samples were mutated within exon 20 (31.6%). In operable breast cancer 1/30 (3.3%) cfDNA sample was mutated in exon 9 and 3/30 (10%) cfDNA samples were mutated in exon 20, while in metastatic breast cancer 2/45 (4.4%) cfDNA samples wer mutated in exon 9 and 9/45 (20%) cfDNA samples were mutated in exon 20. PIK3CA mutations were not detected in any of the 12 cfDNA samples from healthy donors, used as negative controls. Conclusions: Using High-Resolution Melting curve Analysis (HRMA) PIK3CA gene mutations were detected in cfDNA circulating in plasma of patients with operable and metastatic breast cancer, but not in plasma of healthy individuals. The prognostic significance of this finding will be evaluated when a longer follow up period will be completed for these patients and a larger number of clinical samples will be analyzed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2088. doi:1538-7445.AM2012-2088