Aim and patient cohort
This prospective study (NCT03381326) was approved by the Institutional Review Board of IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori,” Meldola, Italy (protocol code: IRST B030). Inclusion criteria were: histology of prostate adenocarcinoma without neuroendocrine differentiation, a progressive disease despite “castration levels” of serum testosterone (< 50 ng/dL), ongoing LHRH analog treatment or prior surgical castration, and evidence of PD during or after treatment with docetaxel. Additional eligibility criteria included an Eastern Cooperative Oncology Group (ECOG) performance status 0–2, adequate cardiac, renal, hepatic, and bone marrow function. PD was defined as either biochemical progression (three consecutive rises in PSA 1 week apart, or radiologic progression (consisting in the appearance of new lesions using the Response Evaluation Criteria in Solid Tumors) . Serum PSA was evaluated within 3 days of beginning therapy and monthly thereafter. Radiographic disease was assessed with the use of computed tomography and bone scan at the time of screening and every 12 weeks on treatment. The study was conducted in accordance with the Declaration of Helsinki and the Good Clinical Practice guidelines of the International Conference of Harmonization. Written informed consent was obtained from all patients.
Study design and blood sampling
We collected blood samples from 100 patients at different time points: pre-treatment, after one cycle treatment (optional), at first radiological evaluation, at the end of treatment/at PD. Peripheral blood samples were collected pre-treatment (baseline) in cfDNA BCT tubes (STRECK, USA), centrifuged at 1800×g for 15 min and plasma aliquots were stored at -80 °C. Two 5-ml peripheral blood samples from each patient were retrieved at different time points in AdnaCollectTM tubes (Qiagen) to enrich and isolate CTC.
DNA isolation and quantification
Plasma DNA was extracted with the QIAamp Circulating Nucleic Acid Kit (Qiagen, Milan, Italy) according to the manufacturer’s instructions, using 1 mL of plasma. Total extracted DNA was quantified by a spectrophotometer (Nanodrop ND-1000, Celbio, Milan, Italy) using 2 μl of DNA.
Digital PCR analysis
AR copy number (CN) analyses were performed by QuantStudio3D digital Polymerase Chain Reaction (dPCR) System (Thermo Fisher Scientific) in a duplex assay using FAM and VIC fluorescent probes. AR CN was evaluated with two assays (AR1: Hs04107225; AR2: Hs04511283) and two reference genes were selected as control genes: RNaseP, TaqMan Copy Number Reference Assay, and AGO1 (Hs02320401), modified with a VIC-labeled probe. DNA samples from three healthy male donors were pooled and used as calibrator. Data were analyzed using QUANT STUDIO 3D ANALYSIS SUITE CLOUD Software (Thermo Fisher Scientific). The average number of copies per reaction microlitres was determined using Poisson distribution. A ratio of target copies and reference copies was measured for each sample, then a ratio between sample and calibrator was calculated. AR CN status is defined as the mean value of the result of the two assays. AR gain status was defined using a cut-point value > 2.01, as previously described .
CTC enrichment and detection
CTC were isolated by the immunomagnetic-based AdnaTest EMT-2/StemCell Select™, targeting EPCAM, EGFR, and ERBB2. This method permits the characterization of CTC and the assessment of potential biomarkers in CTC, even though it does not allow CTC enumeration or morphology.
The cell lysates derived were stored at − 20 °C no longer than 2 weeks, then proceeding with mRNA-isolation by AdnaTest EMT-2/StemCell Detect™ (both Qiagen) according to manufacturer’s instructions. Oligo (dT)25-coated beads allow mRNA isolation from the lysate of pre-enriched CTC. cDNA was obtained from reverse transcription using Sensiscript Reverse Transcriptase Kit™ (Qiagen), as previously described .
Gene expression profiling
List of target genes
We selected 17 cancer-related assays from the TATAA GrandPerformance CTC Assay Panel, a customizable gene panel that allows single-cell expression profiling that was run at TATAA Biocenter. Biomarkers’ choice was based on their role in prostate cancer progression and metastasis, steroid synthesis and signaling, stemness, EMT process, neuroendocrine differentiation. All samples were profiled in duplicates for expression of AR-V7, AKR1C3, AKT2, ALDH1, AR, EPCAM, PSMA, MDK, PARP, MRP1, PI3KCA, POU5F1, PSCA, TUBB3, VIM, ACTB, and HPRT1. This gene expression panel is composed of 17 genes, of which ACTB was used as a control gene. The limit of quantification (LoQ) for the assays in the panel ranged from 20 to 200 copies/reaction. LoQ was calculated as the concentration of the last standard point before the RSDr (relative standard deviation of replicates) is > 35% based on copy number. Regarding specificity, capillary gel electrophoresis was performed during assays validation in order to identify that the PCR product had the correct length in bp (+/− 10 bp) as expected, indicating that the specific target was amplified.
CTC positivity for the examined patient cohort was defined as the expression of at least 1 of the following 12 biomarkers: AR-V7, AKR1C3, AKT2, ALDH1, AR, EPCAM, PSMA, MDK, PIK3CA, PSCA, TUBB3, VIM. These biomarkers were selected based on literature data supporting their capacity to identify CTC as positive in prostate cancer or confirming their role in prostate cancer or in EMT [28,29,30].
The samples were preamplified using TATAA PreAmp Primer Mix (TATAA Biocenter AB) and TATAA PreAmp GrandMaster® Mix (Cat. No. #TA05, TATAA Biocenter AB). Preamplification was performed in a thermocycler (T100, BioRad). No template controls (preAmp NTC) and human genomic DNA samples (0.5 ng/μl, TATAA Biocenter) were included.
The preamplified samples were 8× diluted according to the recommendation for the TATAA PreAmp GrandMaster® Mix. The diluted preamplified samples were analyzed for the 17 genes selected from the GrandPerformance CTC Assay Panel (TATAA Biocenter AB) and the ValidPrime™ assay (TATAA Biocenter AB). The qPCR analysis was performed using TATAA Probe GrandMaster® Mix (TATAA Biocenter AB). No template control and preAmp NTC were included, as described elsewhere . Cycle of quantification (Cq) values above 35 were treated as “off scale data” and replaced with a Cq of 50. The raw data (averaged Cq-values) were examined and corrected for genomic DNA contamination using the GenEx software (MultiD Analyses AB). The averaged Cq-values corrected with more than 1 Cq-value were removed from the analysis due to large-scale genomic DNA contamination. The expression data were normalized to the ACTB control gene for all the testing genes due to its stability and abundance in most of the samples analyzed. The Cq values were converted to relative quantities and transformed to log base 2 scale.
Biomarker values evaluated at baseline and over time were summarized among all patients for whom at least one blood sample was available using descriptive statistics (absolute and relative frequency for categorical biomarkers whereas means ± standard deviation or median and interquartile range for continuous biomarkers).
Relationship among clinical characteristics and the value of each biomarker at baseline was investigated using chi-square test or Fisher’s test, as appropriate.
OS was calculated from the date of the start of cabazitaxel to death or last follow-up. Progression-free survival (PFS) was calculated as the time between the start of cabazitaxel and the first date of progression or death, whichever comes first, or last tumor evaluation.
Time-to-event outcomes (i.e. PFS and OS) were evaluated using the Kaplan-Meier method. Univariate and multivariable Cox regression models were used to estimate the hazard ratio (HR) and relative 95% confidence intervals (95% CI). To identify prognostic factors for OS and PFS, multivariable Cox regression model with stepwise backward elimination method were performed.
The effect of the interaction between AR-V7 expression and initial cabazitaxel dose on OS was evaluated using Cox regression model including AR-V7 expression, initial cabazitaxel dose, and initial dose-by-AR-V7 expression.
The impact of change in biomarkers at various landmark times on survival outcomes was assessed by landmark analyses. Patients with early PD/death before the landmark times were excluded. For these analyses, PFS and OS times were measured from the landmark times to these survival outcomes. The landmark times were at 1 month and 3 months of treatment.
Logistic regression analysis was performed to estimate odds ratio (OR) and 95% CI of the association between biomarkers and PSA response and early PD, defined as PD occurring within the first three months of treatment.
All tests were two sided, and p< 0.05 was considered significant. Statistical analysis was conducted using SAS Statistical Software release 9.4 (SAS Institute, Cary, NC, USA).