EURURO Vol. 72 No. 4

510 656

1. Introduction
Although prognosis for localised prostate cancer patients
following radical prostatectomy is very good, 4–25%
(dependent upon disease stage and use of population
prostate-specific antigen [PSA] screening) will develop
metastatic disease within 15 [38_TD$DIFF]years . In addition, patients
with low- and some intermediate-risk prostate cancers are
best treated by active surveillance; however, there is clinical
uncertainty about progression in this population Pro-
gression in low/intermediate risk may be due to a more
biologically aggressive genotype of primary tumours, whilst
in clinically higher risk groups there may be undetected
micrometastatic disease at presentation This could be
treated by adjuvant approaches including pelvic radiothera-
py extended lymph node dissection adjuvant
hormone therapy , or chemotherapy
Presently, metastatic risk is estimated from histopatho-
logic grade (Gleason score [GS] and clinical grade grouping),
tumour stage, and presenting PSA level. These prognostic
factors have limitations;15% of lower-grade prostate cancer
patients (Gleason 7) experience disease recurrence
whereas 74–76% of higher-grade patients (Gleason >7)[39_TD$DIFF] do
not develop metastatic disease following surgery For
Gleason 7 tumours, dominant lesion grade affects prognosis,
40% of Gleason 4 + 3 patients developing recurrence by
5 [38_TD$DIFF]years compared with 15% for Gleason[40_TD$DIFF] 3 + 4 Clearly,
there is a need to identify additional prognostic factors to
guide adjuvant treatment. Current approaches can broadly be
classified as mathematical risk models using clinical factors
such as Cancer of the Prostate Risk Assessment (CAPRA)
and CAPRA-surgery (CAPRA-S) scoring, or biomarkers
measured from tumour tissue. Regarding biomarkers,
researchers have taken immunohistochemical approaches
such as high Ki67 expression or PTEN loss to indicate
metastatic potential Others have used multiplexing
approaches where a gene expression or proteomic
signature has been trained against known outcomes to
predict high- and low-risk disease using archived material.
It is recognised that malignancies originating from the
same anatomical site can represent different molecular
entities We hypothesised that a unique molecular
subgroup of primary prostate cancers may exist that has a
gene expression pattern associated with metastatic disease.
We took an unsupervised hierarchical clustering approach
using primary localised prostate cancer, primary prostate
cancer presenting with concomitant metastatic disease,
lymph node metastasis, and normal prostate samples to
identify a novel ‘‘metastatic subgroup’’. A 70-transcript
signature (metastatic assay) was developed using this
approach and independently validated in a cohort of radical
prostatectomy samples for biochemical and metastatic
recurrence.
2. Patients and methods
2.1. Study design
Study design followed the reporting recommendations for tumour
marker prognostic studies (REMARK) guidelines as outlined in the
criteria checklists (Supplementary Table 1 and Appendix A) and REMARK
study design diagram (Supplementary Fig. 1).
2.2. Patients
Formalin-fixed paraffin-embedded (FFPE) sections from 126 samples
(70 primary prostate cancer specimens from radical prostatectomy
resections including those with known concomitant metastases, 31 met-
astatic disease in lymph nodes, and 25 histologically confirmed normal
prostate samples that did not display hypertrophy, sourced from bladder
resections) were collected from the University of Cambridge and the
Institute of Karolinska for molecular subgroup identification (Supple-
mentary Table 2). A secondary training dataset of 75 primary resection
samples was collected, of which 20were profiled in duplicate, to aid in the
selection of the final signature length (Supplementary Table 3). For
independent in silico validation, three public datasets were identified
GSE25136 (n = 79; Supplementary Table 4), GSE46691
(n = 545; Supplementary Table 5), and GSE21034 (n = 126; Supplemen-
tary Table 6). A total of 322 FFPE prostatectomy samples from four sites
were collected for independent validation of the assay (Supplementary
Table 7). Biochemical recurrence was defined as a [41_TD$DIFF]post-prostatectomy rise
in PSA of >0.2 ng/ml followed by a subsequent rise. Metastatic recurrence
was defined as radiologic evidence of any metastatic disease, including
lymph node, bone, and visceral metastases. Inclusion criteria were T1a–
T3c NXM0 prostate cancers treated by radical prostatectomy, no previous
systemic adjuvant or neoadjuvant treatment in [42_TD$DIFF]non-recurrence patients,
and at least 3-yr follow-up. Ethical approval was obtained from East of
England Research Ethics Committee (Ref: 14/EE/1066).
2.3. Metastatic subgroup and assay discovery
The 126 discovery samples were analysed for gene expression using a cDNA
microarray platform optimised for FFPE tissue. Unsupervised hierarchical
clustering, an unbiased statistical method to discover structure in data, was
applied to the gene expression profiles. Genes were selected using variance-
intensity ranking and then an iterative procedure of clustering with
different gene lists to determine the optimal set for reproducibility. Data
matrices were standardised to median gene expression and agglomerative
two-dimensional hierarchical clustering was performed, using Euclidean
risk of biochemical and metastatic recurrence superior to either model alone
(HR = 2.67 [1.90–3.75]; p < 0.0001 and HR = 7.53 [4.13–13.73]; p < 0.0001, respectively).
The retrospective nature of the study is acknowledged as a potential limitation.
Conclusions: The metastatic assay may identify a molecular subgroup of primary prostate
cancers with metastatic potential.
Patient summary: The metastatic assay may improve the ability to detect patients at risk of
metastatic recurrence following radical prostatectomy. The impact of adjuvant therapies
should be assessed in this higher-risk population.
# 2017 European Association of Urology. Published by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license .
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