Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen

Abstract

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens.

Keywords: breast cancer; cell-free DNA; disease progression; tamoxifen therapy; targeted next generation sequencing.

Conflict of interest statement

None of the authors has a conflict of interest.

Figures

Figure 1. Study design and discovered DNA changes

Targeted ion-PGM (re-)sequencing was performed on DNA isolated from primary tumors and blood specimens from 10 metastatic breast cancer patients who received tamoxifen as first-line therapy. Cell-free DNA (cfDNA) was isolated from 400 μl serum taken at start (Ss), during therapy (St) and at disease progression (Sp). Analysis revealed 12 biomarkers including 9 single nucleotide variants (SNVs) detected at progression and in primary tumor but not in all preceding blood specimens. The SNVs originating from the primary tumor are presented in red when only seen at Sp, in green when seen at Ss and Sp, and in blue when seen at St and Sp.

Figure 2. cfDNA missense mutations and disease development

The AKAP9 and PIK3CA mutations of patient 1 were evaluated in blood specimens during the course of disease. Sera collected five years after clinical diagnosis of breast cancer were evaluated by ion-PGM resequencing and for PIK3CA in duplicate by digital PCR (dPCR). The p.H1047R mutation was observed at low magnitude in blood taken five years after diagnosis of primary disease and already two years before radiological diagnosis of metastatic lesions. Although sometimes low numbers of PIK3CA mutant copies were detected, all were independently observed after ion-PGM resequencing and in two separate digital PCR reactions. All proportions except for the sample at 5y11m were above the limit of detection.