Frequent detection of PIK3CA mutations in single circulating tumor cells of patients suffering from HER2-negative metastatic breast cancer

Abstract

Modern technologies enable detection and characterization of circulating tumor cells (CTC) in peripheral blood samples. Thus, CTC have attracted interest as markers for therapeutic response in breast cancer. First studies have incorporated CTC analyses to guide therapeutic interventions and stratification of breast cancer patients. Aim of this study was to analyze characteristic features of CTC as biomarker for predicting resistance to HER2-targeted therapies. Therefore, CTC from metastatic breast cancer patients with HER2-negative primary tumors screened for the prospective randomized phase III trial DETECT III were explored for their HER2 status and the presence of PIK3CA mutations. Detection and characterization of HER2 expression of CTC were conducted with the CellSearch(®) system. Fifteen of 179 CTC-positive patients (8.4%) contained ≥1 CTC with strong HER2 expression. Genomic DNA from individual CTC isolated by micromanipulation was propagated by whole genome amplification and analyzed for PIK3CA mutations in exons 9 and 20 by Sanger sequencing. One or more CTC/7.5 mL were detected in 179/290 patients (61.7%). In 109 patients (34.8%), ≥5 CTC/7.5 mL were found. We detected at least one CTC with the mutation p.E542K, p.E545K, p.H1047R, p.H1047L or p.M1043V in 12/33 patients (36.4%). Thirty six of 114 CTC (31.6%) harbored one of these mutations. CTC in individual patients exhibited heterogeneity concerning PIK3CA mutations and HER2 expression. In conclusion, clinically relevant genomic aberrations such as mutations in the hotspot regions of exon 9 and 20 of the PIK3CA gene can be detected in single CTC and might provide insights into mechanisms of resistance to HER2-targeted therapies.

Keywords: Circulating tumor cells; HER2; Metastatic breast cancer; PIK3CA.

Figure 1

Analysis of HER2 expression with the CellSearch® system. A: CTC 1–7 display weak to moderate intensity of HER2 immunofluorescence and no HER2 gene amplification applying FISH (B). Only CTC 8 and 9 (from another study) exhibiting strong intensity of HER2 immunofluorescence present with HER2 gene amplification detected by FISH. C: Strong intensity of HER2 immunofluorescence displayed by BT474 breast cancer cell line cells characterized by HER2 gene amplification (D).

Figure 2

Hotspot mutations of the PIK3CA gene in exon 9 (nucleotide exchanges c.1624 G > A (A) or c.1633 G > A (B) leading to amino acid replacements of glutamic acid to lysine p.E542K and p.E545K, respectively) or exon 20 (c.3127 A > G (C), c.3140 A > G (D) or A > T (E) leading to replacement of methionine by valine p.M1043V or histidine by arginine or leucine p.H1047R or p.H1047L, respectively).