A four-group urine risk classifier for predicting outcomes in patients with prostate cancer

Abstract

Objectives: To develop a risk classifier using urine-derived extracellular vesicle (EV)-RNA capable of providing diagnostic information on disease status prior to biopsy, and prognostic information for men on active surveillance (AS).

Patients and methods: Post-digital rectal examination urine-derived EV-RNA expression profiles (n = 535, multiple centres) were interrogated with a curated NanoString panel. A LASSO-based continuation ratio model was built to generate four prostate urine risk (PUR) signatures for predicting the probability of normal tissue (PUR-1), D'Amico low-risk (PUR-2), intermediate-risk (PUR-3), and high-risk (PUR-4) prostate cancer. This model was applied to a test cohort (n = 177) for diagnostic evaluation, and to an AS sub-cohort (n = 87) for prognostic evaluation.

Results: Each PUR signature was significantly associated with its corresponding clinical category (P < 0.001). PUR-4 status predicted the presence of clinically significant intermediate- or high-risk disease (area under the curve = 0.77, 95% confidence interval [CI] 0.70-0.84). Application of PUR provided a net benefit over current clinical practice. In an AS sub-cohort (n = 87), groups defined by PUR status and proportion of PUR-4 had a significant association with time to progression (interquartile range hazard ratio [HR] 2.86, 95% CI 1.83-4.47; P < 0.001). PUR-4, when used continuously, dichotomized patient groups with differential progression rates of 10% and 60% 5 years after urine collection (HR 8.23, 95% CI 3.26-20.81; P < 0.001).

Conclusion: Urine-derived EV-RNA can provide diagnostic information on aggressive prostate cancer prior to biopsy, and prognostic information for men on AS. PUR represents a new and versatile biomarker that could result in substantial alterations to current treatment of patients with prostate cancer.

Keywords: #PCSM; #ProstateCancer; active surveillance; biomarker; cell free; liquid biopsy; urine.

Figure 1

(A) Prostate urine risk (PUR) profiles (PUR‐1, green; PUR‐2 , blue; PUR‐3, yellow; PUR‐4, red) for the training cohort, grouped by D'Amico risk group and ordered by ascending PUR‐4 score. Horizontal lines indicate where the PUR thresholds lie for: 1° PUR‐1 (green, 0.342), 2° PUR‐1 (purple, 0.297), 1° PUR‐4 (red, 0.476), 2° PUR‐4 (orange, 0.219) and the crossover point between PUR‐1 and PUR‐4 (black, 0.123 both PUR‐1 and 4). (B) PUR profiles in the test cohort. (C) Examples of samples with primary PUR signatures, where coloured circles indicate the primary PUR signal for that sample: 1° PUR‐1 (green), 1° PUR‐2 (blue), 1° PUR‐3 (yellow), 2° PUR‐4 (orange) and 1° PUR‐4 (red). The sum of all four PUR signatures in any individual sample is 1, i.e. PUR‐1 + PUR‐2 + PUR‐3 + PUR‐4 = 1. (D) The outline of the four PUR signatures for all samples, ordered in ascending PUR‐4 (red) to illustrate where 1°, 2° and the 3° crossover point of PUR‐1 and PUR‐4 lies.

Figure 2

(A and B) Boxplots of prostate urine risk (PUR) signatures in samples categorized as no evidence of cancer (NEC [n = 62, training; n = 30, test]) and D'Amico risk categories; (L – low‐: n = 89, training, n = 45, test; I – intermediate‐: n = 131, training, n = 69, test; and H – high‐risk, n = 61, training, n = 27, test) in (A) the training and B) the test cohort. Horizontal lines indicate where the PUR thresholds lie for: 1° PUR‐1 (green), 2° PUR‐1 (purple), 1° PUR‐4 (red), 2° PUR‐4 (orange). (C and D) Receiver‐operating characteristic (ROC) curves of PUR‐4 and PUR‐1 predicting the presence of significant (D'Amico intermediate‐ or high‐risk) prostate cancer prior to initial biopsy in (C) the training and (D) the test cohort. Coloured circles indicate the specificity and sensitivity, respectively, of thresholds along the ROC curve that correspond to the indicated PUR‐4 thresholds, equivalent to: red – 1° PUR‐4, orange – 2° PUR‐4, purple – equivalent to 2° PUR‐1, green – equivalent to 1° PUR‐1.

Figure 3

Area under the curve (AUC) for prostate urine risk (PUR)‐4 predicting the presence/absence of Gleason score (GS) ≥7 on initial biopsy in the training and test cohorts (A and B, respectively) and GS ≥4 + 3 in the training and test cohorts (C and D, respectively). Coloured circles indicate the specificity and sensitivity, respectively, of thresholds along the receiver‐operating characteristic curve that correspond to the indicated PUR‐4 thresholds, equivalent to: red – 1° PUR‐4, orange – 2° PUR‐4, purple – equivalent to 2° PUR‐1, green – equivalent to 1° PUR‐1.

Figure 4

Decision‐curve analysis (DCA) plot depicting the standardized net benefit of adopting prostate urine risk (PUR)‐4 as a continuous predictor for detecting significant cancer on initial biopsy, when significant is defined as: D'Amico risk group of intermediate or greater (teal), Gleason score (GS) ≥3 + 4 (orange) or GS ≥4 + 3 (red). To assess benefit in the context of cancer arising in a non‐PSA screened population of men we used data from the control arm of the Cluster Randomized Trial of PSA Testing for Prostate Cancer (CAP) study 29. Bootstrap analysis with 100 000 resamples was used to adjust the distribution of Gleason grades in the Movember cohort to match that of the CAP population. For full details see Methods.

Figure 5

(A) Prostate urine risk ( PUR) profiles of patients on active surveillance (AS) who met the clinical criteria, not including multiparametric MRI criteria, for progression (n = 23) or not (n = 49) 5 years after urine sample collection. Progression criteria were either: PSA velocity >1 ng/mL per year or Gleason score ≥4 + 3 or ≥50% cores positive for cancer on repeat biopsy. PUR signatures for progressed vs non‐progressed samples were significantly different for all PUR signatures (P < 0.001, Wilcoxon rank‐sum test). Horizontal line colour indicates the thresholds for PUR categories described in: (B) Kaplan–Meier plot of progression in AS patients with respect to PUR categories described by the corresponding colours ( green – 1° and 2° PUR‐1, blue – 3° PUR‐1, yellow – 3° PUR‐4, orange – 2° PUR‐4, red – 1° PUR‐4), and the number of patients within each PUR category at the given time intervals in months from urine collection. (C) Kaplan–Meier plot of progression with respect to the dichotomized PUR thresholds described by the corresponding colours (green – PUR‐4 < 0.174, red – PUR‐4 ≥0.174) and the number of patients within each group at the given time intervals in months from urine collection.