Cell-Free DNA Variant Sequencing Using Plasma and AR-V7 Testing of Circulating Tumor Cells in Prostate Cancer Patients

Abstract

Prostate cancer (PCa) is the second most common malignant cancer and is a major cause of morbidity and mortality among men worldwide. There is still an urgent need for biomarkers applicable for diagnosis, prognosis, therapy prediction, or therapy monitoring in PCa. Liquid biopsies, including cell-free DNA (cfDNA) and circulating tumor cells (CTCs), are a valuable source for studying such biomarkers and are minimally invasive. In our study, we investigated the cfDNA of 34 progressive PCa patients, via targeted sequencing, for sequence variants and for the occurrence of CTCs, with a focus on androgen receptor splice variant 7 (AR-V7)-positive CTCs. The cfDNA content was associated with overall survival (OS; p = 0.014), disease-specific survival (DSS; p = 0.004), and time to treatment change (TTC; p = 0.001). Moreover, when considering all sequence variants grouped by their functional impact and allele frequency, a significant association with TTC (p = 0.017) was observed. When investigating only pathogenic or likely pathogenic gene variants, variants of the BRCA1 gene (p = 0.029) and the AR ligand-binding domain (p = 0.050) were associated with a shorter TTC. Likewise, the presence of CTCs was associated with a shorter TTC (p = 0.031). The presence of AR-V7-positive CTCs was associated with TTC (p < 0.001) in Kaplan-Meier analysis. Interestingly, all patients with AR-V7-positive CTCs also carried TP53 point mutations. Altogether, analysis of cfDNA and CTCs can provide complementary information that may support temporal and targeted treatment decisions and may elucidate the optimal choice within the variety of therapy options for advanced PCa patients.

Keywords: CTCs; cfDNA; prognosis; prostate cancer; sequence variants.

Figure 1

Kaplan–Meier analysis: association of pretreatment–treatment groups with prognosis (OS, DSS, TTC).

Figure 2

Kaplan–Meier analysis: association between cfDNA concentration and prognosis (OS, DSS, and TTC). A higher cfDNA concentration (> 17.93 ng/mL) was associated with a shorter OS (p = 0.014), DSS (p = 0.004), and TTC (p = 0.001).

Figure 3

Kaplan–Meier analysis: association between BRCA1 sequence variants and time to treatment change (TTC). Patients with BRCA1 likely pathogenic and pathogenic sequence variants showed a shorter TTC (p = 0.029).

Figure 4

Kaplan–Meier analysis: association between MUC16 likely pathogenic sequence variants and OS or time of metastasis to last follow-up. Patients with likely pathogenic variants in the MUC16 gene showed a longer OS (p = 0.028) and a longer survival time after diagnosis of metastasis (p = 0.025).

Figure 5

Kaplan–Meier analysis: AR-LBD mutations are associated with survival after blood sampling and TTC. Patients with AR-LBD mutations showed a shorter survival after blood sampling (p = 0.036) and, as a trend, a shorter TTC (p = 0.050).

Figure 6

Heatmap variant score. Hierarchical clustering revealed three distinct clusters. The variant score of individual genes (right hand side) and for all individual samples (bottom lane) was clustered using Euclidian distance and average linkage. Hierarchical clustering revealed three distinct clusters marked by the color bars on the top with Group 1 in light blue, Group 2 in green and Group 3 in red.

Figure 7

Association of sequence variant groups with TTC. The three sequence variant groups showed a significant association with TTC (p = 0.017).

Figure 8

Kaplan–Meier analysis: association of CTCs or AR-V7-positive CTCs with time to treatment change (TTC). The presence of CTCs or AR-V7-positive CTCs was associated with a shorter TTC (p = 0.031 or p < 0.001).