AR-V7 Positivity Varies with Circulating Tumor Cell (CTC) Enrichment and Novel CTC Enrichment Is Able to Reduce AR-V7 False Positivity

Background: The AR-V7 splice variant is a cause of castration-resistant prostate cancer (CRPC). However, testing for the presence of AR-V7 by RT-PCR shows AR-V7 positivity in healthy individuals. We hypothesized that the positivity reflected contamination by hematopoietic cells. We tried a novel CTC enrichment instrument using Celsee® to clear hematopoietic cells. Methods: We tested whole blood or Celsee-enriched samples for AR-V7 by RT-PCR and included samples from 41 CRPC patients undergoing sequential therapy. We evaluated the associations between AR-V7 status and clinical factors. We evaluated factors affecting AR-V7 positivity. Results: AR-V7 positivity was lower in Celsee-enriched than in whole blood specimens. AR-V7 and clinical factors did not predict the therapy effectiveness. We found no significant differences in the effectiveness of Enz/Abi upon AR-V7 evaluation. All AR-V7 positive patients had resistance to Enz/Abi. DTX, CBZ, and Radium223 treatment showed no significant difference in the treatment effectiveness, regardless of AR-V7 presence. AR-V7 was more frequently positive than EOD2 in cases with bone metastases. Conclusion: Celsee® CTC enrichment suppresses the AR-V7 false positivity. All AR-V7 positive patients presented resistance to Enz/Abi. DTX, CBZ, and Radium223 were effective and remained treatment options. AR-V7 positivity should progressively appear in patients with advanced bone metastases.

low specificity for the test system (13). Hematopoietic cells expressed prototype AR mRNAs (14), and they may also express AR-V7 (for example, in leukocytes) (15).
Given the differing sensitivity and specificity of AR-V7 assessment tests depending on the test system, we considered the following two points to be important when assessing AR-V7 positivity: one was to exclude hematopoietic cells as much as possible during CTC enrichment, the second one was to target AR-V7 mRNA from CTCs during detection.
Celsee® (Celsee Diagnostics, Plymouth, MI) is a microfluidic device that can be used for CTC enrichment. Compared with CellSearch ® , which is the only system approved by the US FDA, the capture rate of Celsee ® for CTCs is high (16). The basic principle is based on the larger size and non-deformability of CTCs compared with those of hematopoietic cells; thus, the system's chamber ensures that small hematopoietic cells escape, whereas larger CTCs get trapped and can be isolated in the chamber. The Celsee® microfluidic device allows for physical CTC enrichment ; its CTC capturing efficiency is greater than 80% and the background of hematopoietic cells contamination in the captured cell population is minimal (17). For CTC identification, we used real-time polymerase chain reaction (RT-PCR) to detect AR-V7 messenger ribonucleic acid (mRNA).
We hypothesized that Celsee® CTC enrichment would suppress AR-V7 false positives.
The first aim of this study was to confirm the difference in the detection rate of AR-V7 between the systems PAXgene® without CTC enrichment and Celsee® CTC enrichment. We used samples from patients with CRPC treated with sequential therapy.
We assumed that analysis of AR-V7 using Celsee would predict the outcome of CRPC treatment, and our second aim was to evaluate the associations between the current treatment efficacy and variables like the presence of AR-V7 and prognostic or clinical factors (PSA levels, AR-V7 is thought to be positive during CRPC treatment. Our third aim was to identify factors affecting AR-V7 positivity by examining the association between prognostic and clinical factors. Table 1 lists baseline characteristics of 41 patients with CRPC. We found AR-V7 positivity in 22 (53.7%) PAXgene®-processed samples and in 9 (22.0%) In Celsee®-processed ones. We confirmed AR-V7 positivity differences between PAXgene® and Celsee®-processed samples from single patients.  for stable disease or response and 16.7 months (13.6) for disease progression (P = 0.005).   Table 3 shows the results of our multivariate analysis. We found no significant differences in  We confirmed the effectiveness of Enz and Abi in past and current treatments of patients with or without AR-V7 positivity. We found no statistically significant differences, but none of the AR-V7-positive patients displayed Enz and Abi treatment effectivity (P = 0.066; Table 4). We examined the effectiveness of DTX, CBZ, and Radium223 with and without AR-V7 positivity and found no statistically significant differences between treatments (P = 0.217; Table 5).   Among the patients with bone metastasis, the Celsee AR-V7-positive/negative ratio was 3 (7.3%)/27 (65.9%) in patients with EOD2 classification or lower, and the Celsee AR-V7 positive/negative ratio was 6 (14.6%)/5 (12.2%) in those with EOD3 or higher (P = 0.006).

Results
Following our multivariate analysis (Table 7) we found no significant PSA differences between AR-V7-positive and -negative samples (HR, 1.004; 95% CI, 1.000-1.005; P = 0.058). The difference in the ratio of patients with EOD2 or lower/patients with EOD3 or higher was confirmed (HR, 0.110; 95% CI, 0.017-0.725; P = 0.022).  (18). He also divided samples into those with higher and lower AR-V7 transcript numbers and showed that patients with more AR-V7 transcripts had a shorter time to treatment failure in the Enz cohort, but not in the Abi cohort (18). With regard to overall survival (OS), He found no significant differences, but they also reported that the time to treatment failure and OS were significantly shorter in patients with positive PSA and higher-AR-V7 expressions than in the others (18). These three reports suggest that when CTC enrichment is not performed, AR-V7 derived from hematopoietic cells affects the analysis results and makes the AR-V7 interpretation difficult regardless of the PCR detection accuracy.
In the Sciarra review, the mCRPC AR-V7 positive rate was reported at 18.3% (range 17.8%-28.8%) (19), and our Celsee AR-V7 positive rate was similar at 22.0%. However, whether the detected AR-V7 is really derived from CTCs, cannot be confirmed by the detection rate. As The JHU method is known as the AR-V7 PCR detection method after CTC enrichment.
CTC enrichment is done with immuno-magnetic beads coated with anti-EpCAM and anti-Her2, and end-product lysates are evaluated on multiplex PCR with AR-V7, AR, PSA, PSMA, and EGFR primers. In the PROPHECY study, Enz and Abi were effective in 6%-11% of AR-V7 positive patients (11). Physically captured leukocytes in the beads may explain these results by reflecting positive-AR-V7 cells other than CTCs.
In the Epic Sciences platform, the effectiveness of Enz and Abi were both 0% in AR-V7 positive patients (11). This platform covers all nucleated blood cells thawed on slides, and the risk of missing CTCs is extremely low. We wanted to know whether AR-V7 can be accurately evaluated by analyzing protein-level antibody responses. AR-V7 antibody has been reported to show nonspecific reactions (21), and we confirmed the presence of false positive responses to anti-AR-V7 antibody (Supplementary appendix figure 1). However, in the Epic Sciences platform, in addition to the usual CTC definition (CTC is cytokeratin positive, CD45 negative, and DAPI positive), captured images were analyzed using an automated algorithm that characterizes each cell using >90 variables. This morphological digital pathology diagnosis may overcome the nonspecificity of the AR-V7 antibody reactions.
Another implication of our results is that AR-V7 positive patients have more bone metastases than those with EOD3, and these patients have more CTCs in their blood. A limitation of our system is its inability to assess the CTC counts. Other limitations of our study include the small number of samples studied. We targeted patients undergoing sequential therapy for CRPC and could not assess OS or progression-free survival. However, other reports have shown that AR-V7 negative patients do not usually have AR-V7 re-evaluations with disease progression after their initial AR-V7 assessment before the beginning of treatments for CRPC. Nakazawa showed that therapeutic interventions modify the AR-V7 status (22). Studying how the sequential therapy affects prognosis depending on the AR-V7 status is important to be able to appreciate the true value of AR-V7 assessments.

Study design
We measured PSA levels and obtained other laboratory tests for monitoring monthly.
Imaging such as CT, MRI, and bone scintigraphy were performed at least every 3-6 months.
We defined stable disease as that in individuals with <50% PSA decrease to <25% PSA increase from a PSA nadir, and disease response as that in individuals with PSA decrease >50% in the absence of radiographic progression. We defined disease progression as radiographic progression or PSA increases ≥25% and ≥2 ng/mL above the nadir or baseline. We used PCWG3 soft tissue and bone scan criteria to assess radiographic progression (23). Time points of taking peripheralblood samples for analysis of AR-V7 were performed on patients already undergoing treatment for CRPC. Treatment selection was at the discretion of the treating physician without AR-V7 status consideration. Laboratory investigators were blinded to the clinical information and patient outcomes.
Blood collection and RNA extraction (Using Celsee®) We placed 5 mL of blood in anticoagulant ethylene-diamine-tetra-acetic acid (EDTA) blood tubes. We treated 4 mL blood samples with RBC lysis buffer (10×) (BioLegend, San Diego, CA, USA) and let them stand for 20 min on ice. Following centrifugation at 326 × g for 5 min at room temperature, we discarded the supernatants and resuspended the isolated cells in 8 mL of 1% BSA/PBS. We ran the samples through Celsee® and collected the cells on the chip in 1 mL × 8 back flushes using 1% BSA/PBS and centrifuged them at 326 × g for 5 min. We used an RNA isolation NucleoSpin® kit (MACHEREY-NAGEL, Düren, Germany) according to the manufacturer's protocol.
(Using Paxgene®) We collected 2.5 mL blood samples into Paxgene® blood tubes and stored them at −80°C. We thawed the blood samples prior to extracting RNA from them using the Paxgene® blood RNA kit (Preanalytix, Hombrechtikon, Switzerland) according to the manufacturer's protocol.

RNA quality control and Reverse transcription
We processed all samples after RNA extraction with a DNase kit (Turbo-DNA-free AM1907; Invitrogen, CA, USA) and then ethanol-precipitated the DNA-free RNA samples with Ethachinmate (NIPPON GENE, JAPAN).
We used the NanoDrop One C system to determine RNA concentrations and absorption at 260 and 280 nm. We only used samples with a 260/280 ratios ≥ 2.0. We performed reverse transcription (RT) with 1 µg of RNA using a High Capacity complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer's protocol.

Polymerase chain reaction
We used the SYBR Green method for our RT-PCR assays. We made cDNA using the KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems, Headquarters, MA, USA) to detect prostate cancerassociated RNA transcripts.
We measured product concentrations using the CFX96 ™ (Bio-Rad, Hercules, CA, USA) instrument and analyzed results with the CFX Maestro ™ software (Bio-Rad). We confirmed that all AR-V7 positive specimens were GAPDH positive.

Basic experiments on Celsee® before clinical trials
We collected 4 mL of whole blood samples from 8 healthy volunteers into EDTA blood tubes.
Subsequently, we performed RNA extraction, RT, and PCR assays on these specimens, and confirmed AR-V7 positivity in 6 cases ( Figure 1). Figure 1： Presence of AR-V7 by PCR in the whole blood of healthy volunteers.