Dataset Information

Analysing the mutational status of PIK3CA in circulating tumor cells from metastatic breast cancer patients.

ABSTRACT: The frequently altered phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is involved in the regulation of cellular processes required for breast carcinogenesis. The aim of the project was to develop a method to identify hotspot mutations in the PIK3CA gene in circulating tumor cells (CTCs) of metastatic breast cancer (metBC) patients. From 44 enrolled CTC-positive metBC patients a total number of 57 peripheral blood samples were analysed by CellSearch(®). Genomic DNA of enriched CTCs was isolated, amplified and analyzed for PIK3CA mutations in exons 9 and 20 which lead to E542K, E545K or H1047R amino acid changes and result in increased PI3K activity. The mutations were detected by using SNaPshot-methodology comprising PCR amplification and single nucleotide primer extension. SNaPshot analysis was established using genomic DNA from different breast cancer cell lines and then successfully transferred to investigate blood samples and single cells. Overall, twelve hotspot mutations in either exon 9/E545K (6/12, 50%) or exon 20/H1047R (6/12, 50%) could be determined within 9 out of 57 (15.8%) blood samples from 7 out of 44 (15.9%) patients; CTC counts ranged from 1 to 9748. PIK3CA variants E542K, E545G and E545A were not detected. Analysing the PIK3CA genotype of CTCs has clinical relevance with respect to drug resistance, e.g. against HER2-targeted therapy. The herein described approach including SNaPshot technology provides a simple method to characterize hotspot mutations within CTCs enriched from peripheral blood and can be easily adopted for analysing further therapeutically relevant SNPs.

SUBMITTER: Schneck H

PROVIDER: S-EPMC5528457 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Analysing the mutational status of PIK3CA in circulating tumor cells from metastatic breast cancer patients.

Schneck Helen H Blassl Christina C Meier-Stiegen Franziska F Neves Rui Pedro RP Janni Wolfgang W Fehm Tanja T Neubauer Hans H

Molecular oncology 20130713 5

The frequently altered phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is involved in the regulation of cellular processes required for breast carcinogenesis. The aim of the project was to develop a method to identify hotspot mutations in the PIK3CA gene in circulating tumor cells (CTCs) of metastatic breast cancer (metBC) patients. From 44 enrolled CTC-positive metBC patients a total number of 57 peripheral blood samples were analysed by CellSearch(®). Genomic DNA of enriched CTCs wa ...[more]

PMID: 23895914

Similar Datasets

Project description:Circulating Tumor Cells (CTCs) represent a "liquid biopsy of the tumor" which might allow real-time monitoring of cancer biology and therapies in individual patients. CTCs are extremely rare in the blood stream and their analysis is technically challenging. The CellSearch(®) system provides the enumeration of CTCs with prognostic significance in patients with metastatic breast cancer (mBC), but it does not allow their molecular characterization, which might be useful to identify therapeutically relevant targets for individualized treatment. Combining the CellSearch(®) and DEPArray™ technologies allows the recovery of single CTCs as a pure sample for molecular analysis. The purpose of the study was to investigate the heterogeneity of PIK3CA mutational status within single CTCs isolated from individual mBC patients. CTCs were enriched and enumerated by CellSearch(®) in blood samples collected from 39 mBC patients. In 20 out of 39 patients enriched samples with ≥5 CTCs were sorted using DEParray™ to isolate single CTCs or pools of CTCs to be submitted to Whole Genome Amplification (WGA) before sequencing analysis. In 18 out of 20 patients, it was possible to perform PIK3CA sequencing on exons 9 and 20. Twelve subjects were wild type (wt) for the PIK3CA gene. PIK3CA status could also be assessed in pools of CTCs in seven of these patients, with consistent wt status found. Six patients (33%) had a PIK3CA mutation identified. In 2 of the six patients, molecular heterogeneity was detected when mutational analysis was performed on more than one single CTC, including one patient with loss of heterozygosity on both single and pooled CTCs, and one patient with three different PIK3CA variants on single CTCs but PIK3CA wt status on pooled CTC samples. In six out of the 18 cases PIK3CA status was also evaluable on a primary tumor sample. In one of the six cases a discordance in PIK3CA status between the primary (wild-type) and the matched CTC (exon 20 mutation) was observed. This study demonstrates the feasibility of a non-invasive approach based on the liquid biopsy in mBC patients. Moreover, our data suggest the importance of characterizing CTCs at the single cell level in order to investigate the molecular heterogeneity within cells from the same patient.

Project description:Liquid biopsy analysis, mainly based on circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides an extremely powerful tool for the molecular profiling of cancer patients in real time. In this study, we directly compared PIK3CA hotspot mutations (E545K, H1047R) in EpCAM-positive CTCs and paired plasma-ctDNA in breast cancer (BrCa). PIK3CA hotspot mutations in CTCs and ctDNA were analyzed using our previously developed highly sensitive (0.05%), specific, and validated assay in plasma-ctDNA from 77 early and 73 metastatic BrCa patients and 40 healthy donors. We further analyzed and directly compared PIK3CA hotspot mutations in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma-ctDNA, in 56 cases of early and 27 cases of metastatic breast cancer, and 16 corresponding primary tumors. In plasma-ctDNA, PIK3CA hotspot mutations were identified in 30/77(39.0%) early and 35/73(47.9%) metastatic BrCa cases; none (0/40, 0%) of the healthy donors' plasma-ctDNA samples were positive. Our direct comparison study in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma-ctDNA from the same blood draws has shown a lack of concordance in early BrCa (27/56, 48.2%), while the concordance in the metastatic setting was higher (18/27, 66.6%). Our results were validated by ddPCR methodology, and the concordance between our assay and ddPCR for PIK3CA E545K hotspot mutation was 30/37 (81.1%). In many cases, PIK3CA hotspot mutations were detected in samples found to be negative for CTCs in CellSearch® . Our data demonstrated for the first time that (a) PIK3CA hotspot mutations are present at high frequencies in CTCs isolated from CellSearch® cartridges and paired plasma-ctDNA both in early and metastatic BrCa, (b) the detection and concordance of PIK3CA hotspot mutations between plasma-ctDNA and CTCs are higher in the metastatic setting, (c) PIK3CA mutational status significantly changes after therapeutic intervention, and (d) PIK3CA mutation detection in CTCs and plasma-ctDNA provides complementary information.

Dataset Information

Analysing the mutational status of PIK3CA in circulating tumor cells from metastatic breast cancer patients.

Publications

Analysing the mutational status of PIK3CA in circulating tumor cells from metastatic breast cancer patients.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets