Cancers | Free Full-Text | Filamin A Is a Prognostic Serum Biomarker for Differentiating Benign Prostatic Hyperplasia from Prostate Cancer in Caucasian and African American Men

By inergency On Feb 8, 2024

This study analyzed a previously published cohort of men whose age ranged between 35 and 92 years old and who had a median age of 63 years and analyzed a specific combination of markers for the investigation of its utility in Caucasian as well as African American men. Within this study cohort, 300 men had BPH (191 CA, 48 AA, and 61 other-race), while 477 men had PCa (281 CA, 139 AA, and 57 other-race) (Table 1). Men with BPH were subjected to at least one biopsy to indicate their PCa-negative status. In contrast, men with PCa were confirmed with biopsy. All men in the analysis cohort had a PSA in the range of 4–10 ng/mL and a negative digital rectal exam (DRE). Additional clinical characteristics of the study cohort are described in Table 1, also being previously published in [4].

In our previous study, we showed that logistic regression using a panel of FLNA, age, and prostate volume performed better than PSA in discriminating between men with PCa and men with BPH. Here, we evaluated five parsimonious logistic regression models comprised of at most two of these features (FLNA alone, PSA alone, age alone, FLNA and age, and PSA and age) and compared their performance to our previous model using FLNA, age, and prostate volume. Models were evaluated by their capability to distinguish PCa (aggressive, i.e., Gleason ≥ 7, as well as comparison of all patients with PCa) from BPH using AUC as a performance metric computed at each model’s optimal cutoff, determined at a sensitivity ≥ 0.9. Finally, model performances were assessed on CA and AA patient subsets separately for each classification analysis.

Quantitation of FLNA

Antibody immobilization. Three mouse monoclonal antibodies, Anti-FLNA 2C12 and Anti-FLNA 3F4, were immobilized using the Thermo Fisher Scientific Pierce Direct IP Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendations, with a few modifications as previously described [12]. In total, 200 µg of each of the antibodies was coupled individually to 200 µL of AminoLink Plus coupling resin and stored at 4 °C until needed.

Immunoprecipitation calibration standard generation. Immunoprecipitation tubes were prepared by aliquoting 5 µL of each of the two antibody-coupled resins into the IP tube (Pierce Direct IP Kit, Thermo Fisher Scientific). The resin was washed twice with 200 µL of an IP lysis/wash buffer. In total, 100 µL of a human serum sample or 100 µL of water (surrogate matrix) was added to each IP tube along with 500 µL of a prepared lysis buffer solution (IP lysis/wash buffer with 1.2X Halt protease cocktail inhibitor; Thermo Fisher Scientific) and 0.5 M EDTA, then incubated overnight at 4 °C with end-over-end mixing. The resin was washed five times with 200 µL of the IP lysis/wash buffer and once with 100 µL of a 1X conditioning buffer. The captured proteins were eluted with 50 µL of an elution buffer with an incubation time of 15 min, and then neutralized with 5 µL of 1 M Tris HCl, pH 9.0 (Teknova, Hollister, CA, USA). The IP eluates from the surrogate matrix were used to prepare P2 (AGVAPLQV) peptide calibration curves by spiking with a P2 synthetic peptide (Genscript, Piscataway, NJ, USA) stock solution (0.2/0.36 µg/mL) followed by serial dilution. P2 calibration standards ranged from 125 pg/mL to 2000 pg/mL. All samples were then subjected to trypsin digestion as described below.

Trypsin Digestion of IP-extracted samples. Trypsin digestion was performed using the Flash Digest Kit (Perfinity Biosciences, West Lafayette, IN, USA) following the manufacturer’s protocol with few modifications. Flash digest tubes were equilibrated to room temperature, and then centrifuged for 1 min at 1500× g and 5 °C. In total, 50 µL of each sample, 25 µL of a digestion buffer (Perfinity Biosciences), and 5 µL of a working internal standard (Thermo Fisher Scientific) solution (P2/P4 10/30 ng/mL) were added to the Flash digest tubes. After vortexing, samples were digested at 70 °C for 20 min in Eppendorf Thermo Mixer C (Eppendorf, Framingham, MA USA). The Flash digest tubes were then centrifuged for 5 min at 1500× g and 5 °C. A 60 µL aliquot of the supernatant was transferred to an LC-MS vial.

LC-MS/MS analysis. MRM analyses were performed on a 6500 QTRAP mass spectrometer (SCIEX) equipped with an electrospray source, a 1290 Infinity UPLC system (Agilent Technologies, Santa Clara, CA, USA), and an Xbridge Peptide BEH300 C18 (3.5 μm, 2.1 mm × 150 mm) column (Waters, Milford, MA, USA). Liquid chromatography was carried out at a flow rate of 400 µL/min, and the sample injection volume was 30 µL. The column was maintained at a temperature of 60 °C. Mobile phase A consisted of 0.1% formic acid (Sigma Aldrich, St. Louis, MO, USA) in water (Thermo Fisher Scientific), and mobile phase B consisted of 0.1% formic acid in acetonitrile (Thermo Fisher Scientific). The gradient with respect to %B was as follows: 0–1.5 min, 5%; 1.5–2 min, 5–15%; 2–5 min, 15%; 5–7.1 min, 15–20%; 7.1–8.1 min, 20–80%; 8.1–9.0 min, 80%; 9.0–9.1 min, 80–5%; and 9.1–16 min, 5%. The instrument parameters for the 6500 QTRAP mass spectrometer were as follows: ion spray voltage of 5500 V, curtain gas of 20 psi, collision gas set to “medium”, interface heater temperature of 400 °C, nebulizer gas (GS1) of 80 psi and ion source gas (GS2) of 80 psi, and unit resolution for both Q1 and Q3 quadrupoles.

PMRM peptide quantitation. A data analysis was performed using Analyst^® software (version 1.6.2, SCIEX, Framingham, MA, USA) and peak integrations were reviewed manually. The calibration curve for the FLNA P2 peptide was constructed by plotting the peak area ratios (analyte/internal standard) versus concentration of the standard with 1/×2 linear least square regression. The regression equations from P2 calibration standards were used to back-calculate the measured P2 concentrations for each QC and unknown sample.