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.

Circulating tumor cells in breast cancer

“…As any other tumor material (surgical specimen, biopsy, fine needle aspirate. ), CTC can be characterized with "omics" analyses: genomics, either focused on copy-number profiles (Kanwar et al, 2015;Neves et al, 2014;Polzer et al, 2014), ERBB2 amplification (Krishnamurthy et al, 2013;Mayer et al, 2011), point mutations (Deng et al, 2014;Fernandez et al, 2014;Markou et al, 2014;Pestrin et al, 2015;Schneck et al, 2013) or on DNA methylation (Chimonidou et al, 2013a(Chimonidou et al, , 2013b(Chimonidou et al, , 2011, transcriptomics (Mostert et al, 2015;Onstenk et al, 2015b;Powell et al, 2012;Sieuwerts et al, 2011) and proteomics. Among proteins of potential therapeutic interest, the estrogen and progesterone receptors (Nadal et al, 2012;Somlo et al, 2011), HER2 (Ligthart et al, 2013), Ki67 (Paoletti et al, 2015) and apoptosis and DNA-repair related proteins (Garcia-Villa et al, 2012;Smerage et al, 2013) have been investigated, together with PDL1 expression on CTC (Mazel et al, 2015) and many other potential biomarkers.…”

Clinical Applications of Circulating Tumor Cells in Pharmacotherapy: Challenges and Perspectives

“…Phosphatidylinositide 3-kinase is a cell membrane signal transduction molecule that supports cell survival and growth, making it a popular therapeutic target (Wong et al, 2010;Akinleye et al, 2013). PIK3CA mutations were identified in CTCs from metastasis breast cancer patients by CellSearch enrichment, DNA extraction, and whole genome amplification (Schneck et al, 2013); therefore, agents that target this pathway, such as everolimus and temsirolimus, are promising therapeutic options (Johnston, 2015).…”

Circulating Tumor Cells: A Window to Understand Cancer Metastasis, Monitor and Fight Against Cancers

“…As PCR is a sensitive method to detect cancer specific genetic alterations, RT-PCR has been used in several recent CTC studies to detect the expression of tumor-related genes, including MUC1 [150], TMPRSS2-ERG fusion gene [151], CK19 [152,153], ERBB2 [51,52,92], EMT related markers [154], or genes associated with drug resistance, such as PIK3CA [155]. Same as FISH, specific primer and target gene selection is required for this analysis.…”