Senologie - Zeitschrift für Mammadiagnostik und -therapie 2016; 13 - A95
DOI: 10.1055/s-0036-1583416

Sequential analysis of circulating tumor cells on genome and protein level: potential regulation of the invasion marker CapG by PIK3CA

M Neumann 1, Y Decker 1, A Franken 1, S Schömer 1, H Schneck 1, T Fehm 1, W Weichert 2, V Endriss 2, H Neubauer 1, D Niederacher 1
  • 1Department of Gynaecology University Hospital Düsseldorf, Research Laboratory, Düsseldorf, Deutschland
  • 2Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, München, Deutschland

Overexpression of macrophage capping protein (CapG) results in increased invasion potential in cancer cells. PI3K driven signal-transduction could lead to an increased CapG expression. Here we show isolated single CTC characterization for the invasion marker CapG and PIK3CA mutations.

Classification of CTCs was done according to CellSearchTM. CTCs were isolated from CellSearch-Cartridge using the CellCelector (ALS, Jena) and transferred onto glass slides for subsequent immune fluorescence detection of CapG. A CapG-Score was established with CapG-negative white blood cells (WBC, CapG-Score 0) and two breast cancer cell lines (BT-20, CapG-Score 1+ and MDA-MB 231, CapG-Score 3+). After CapG quantification CTCs were isolated again and deposited in PCR tubes for whole genome amplification (WGA) with Ampli1 (Silicon Biosystems, Italy). Quality of WGA products was determined and targeted next generation sequencing was done with IonTorrent® AmpliSeq™ Custom Panels (Silicon Biosystems) with a focus on mutations in PIK3CA gene.

In 59 CTCs isolated from 13 breast cancer patients expression of CapG was weak in 40% (Score 1+), strong in 37% (Score 2+) and very strong in 15% (Score 3+). In five of 7 CTCs from 2 patients with CapG-Scores of 2+ and 3+ mutation analysis detected the hot spot point mutation in exon 9 in the PIK3CA gene (E545K).

Isolation of single CTCs using the CellCelector micromanipulator enables sequential characterization of single cells to determine functional relevance of genomic changes on the protein level.