In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells

Abstract

BACKGROUND Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. METHODS We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. RESULTS In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1–76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. CONCLUSIONS Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

Direct comparison of size-dependent versus EpCAM-dependent CTC enrichment at the gene expression and DNA methylation level in head and neck squamous cell carcinoma

“…These workflows were combined with Parsortix system enabling the recovery of higher quality RNA 31 . El-Heliebi et al compared three different CTC isolation systems for gene expression and DNA mutation analysis in CTCs of prostate cancer patients and showed also differences 32 . The incorporation of microfluidics into CTC isolation is now emerging for clinical applications 33 .…”

Liquid biopsy in pancreatic ductal adenocarcinoma: current status of circulating tumor cells and circulating tumor DNA

“…applied KRAS as a marker for CTC enumeration and molecular characterization. They used an in vivo isolation of CTCs (GILUPI CellCollector ® ) directly from the vein of patients and applied signal amplification of in situ padlock probes via rolling‐circle amplification: 47% (7/15) of patients were CTC‐positive (range, 1–3 CTCs/patient), and 40% (6/15) of patients had KRAS mutant CTCs (El‐Heliebi et al ., ).…”

“…Interestingly, El-Heliebi et al applied KRAS as a marker for CTC enumeration and molecular characterization. They used an in vivo isolation of CTCs (GILUPI CellCollector Ò ) directly from the vein of patients and applied signal amplification of in situ padlock probes via rolling-circle amplification: 47% (7/15) of patients were CTC-positive (range, 1-3 CTCs/patient), and 40% (6/15) of patients had KRAS mutant CTCs (El-Heliebi et al, 2018). With regard to the enrichment strategies, size-based filtering strategies exhibited higher sensitivity in isolating CTCs compared with EpCAM-based approaches in patients with metastatic or inoperable pancreatic cancer: ISET and CellSearch Ò detected CTCs in 88.9% (38/50) and in 39.6% (21/53) of patients, respectively (Khoja et al, 2012).…”

Liquid biopsies

“…25 Padlock probe technology that is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. 72 Recently, it was also shown that a biomarker panel containing CTC number and lactate dehydrogenase (LDH) levels was a surrogate for survival at the individual patient level in a clinical trial of abiraterone acetate plus prednisone vs prednisone alone for patients with metastatic CRPC. 73 Analysis of CTC gene expression revealed a clinically prognostic "liquid biopsy" signature in patients with metastatic castrate-resistance PCa.…”