SUPPLEMENTARY INFORMATION
Supplementary Table 1: Subject enrollment by study site
Site
Location
N
Associated Medical Professionals
Syracuse, NY
Associated Medical Professionals
Oneida, NY
9
Columbia Medical Center
New York, NY
8
Delaware Valley Urology
Voorhees, Mt. Laurel and Sewell,
NJ
Integrated Medical Professionals
Elmont, NY
40
Integrated Medical Professionals
Garden, City
9
Integrated Medical Professionals
West Nyack, NY
31
University of Michigan
Ann Arbor, MI
29
Urology Clinics of North Texas
Dallas, TX
99
Urology Specialists, PC
Middlebury, CT
20
Urology Specialists
Las Vegas, NV
23
University Urology Associates
New York, NY
17
36
132
RNA EXTRACTION AND cDNA PREPARATION
Using the Urine Clinical Sample Concentrator Kit, exosomes are isolated from the urine with the
internal control by ultracentrifugation. RNA extraction is performed according to the
manufacturer’s instructions. The final eluted RNA was used for reverse transcription
RNA (14 µl) was reverse transcribed using random primers included in the Superscript VILO kit
(Life Technologies, Carlsbad, CA) in a total volume of 24 µl, in a modification of the
manufacturer’s procedure. A negative (water) and positive control (Human Universal Reference
Total RNA, Clontech, Mountain View, CA) were included with each batch of samples. The
following was added to the qPCR reaction mix (TaqMan® Fast Universal PCR Master Mix, Life
Technologies): 2 µl of cDNA, 0.5 U of uracil-N-glycosylase (Roche) to eliminate potential
amplicon contaminates, primers, and probes. The sequences and concentrations of primers and
probes are provided in Supplementary Information Table 2. Probes for all markers span the
junctions of the amplified exons so that unspliced mRNA is not detected. The ERG primers,
which target exons 10 and 11, were designed to detect wildtype ERG and most fusion partners,
including the most common TMPRSS2-ERG [21].
Real time qPCR was performed using the following program on the Rotor-Gene Q System
(Qiagen, Venlo, Netherlands): 50°C for 2 min, 95 °C for 5 min, followed by 40 cycles of (95°C for
5 s, 60°C for 10 s).
Supplementary Table 2: Primer and probe sequences and concentrations
Primer/
Probe Name
Final Conc.
(µM)
Sequence
Ref Seq and Nucleotide Positions
ERG Forward
GCGTCCTCAGTTAGATCCTTATCAG
Ref Seq NM_182918.3, Pos. 982-1006
0.5
ERG Reverse
CTGGCCACTGCCTGGATT
Ref Seq NM_182918.3, Pos. 1054-1037
1.5
ERG Probe
5‘6FAMCTTGGACCA/ZEN/ACAAGTAGCCGCCTTGC3‘IABkFQ
Ref Seq NM_182918.3, Pos. 1010-1035
0.6
SPDEF Forward
CCACCTGGACATCTGGAAG
Ref Seq NM_012391.2, Pos. 1046-1064
0.1
SPDEF Reverse
AATCGCCCCAGGTGAAGT
Ref Seq NM_012391.2, Pos. 1106-1089
1.0
SPDEF Probe
5‘6-FAMCGGCCTGGA/ZEN/TGAAAGAGCG3‘IABkFQ
Ref Seq NM_012391.2, Pos. 1069-1087
0.3
PCA3 Forward
GCACATTTCCAGCCCCTTTA
Ref Seq NR_015342.1, Pos. 242-261
0.1
PCA3 Reverse
GGCATTTCTCCCAGGGATCT
Ref Seq NR_015342.1, Pos. 319-300
0.5
PCA3 Probe
5‘6FAMCACACAGGA/ZEN/AGCACAAAAGGAAGC3‘IABkFQ
Ref Seq NR_015342.1, Pos. 272-295
0.35
STATISTICAL ANALYSIS
Performance (sensitivity, specificity, positive predictive value [PPV], and negative predictive
value [NPV]) of the binary EXO106 Score for prediction of biopsy result (negative versus positive
for any cancer or high-grade cancer [Gleason Score ≥7]) was assessed by 2x2 contingency tables
with confidence intervals (CI; Wald-intervals) computed from standard errors (SE), assuming a
binomial proportion [1]. An EXO106 Score of 10 was used to discriminate biopsy-positive from
biopsy-negative results. The sample size was chosen based on prior studies which identified a
positive biopsy rate of 45-50% with a 20-25% Gleason score >7 along with an intended use
group of 70% which is sufficient to evaluate AUC and NPV assay performance in the current
enrolled population.
Improvement of the predictive performance of SOC parameters by the EXO106 Score was
assessed as follows: SOC parameters alone (serum PSA levels, family history of PCa, age, and
race) as well as SOC parameters together with EXO106 were modeled by logistic regression to
predict the binary outcome of any cancer or high-grade disease in initial biopsy samples. A
binary cut-point was chosen on the linear predictor scores of both models with and without
EXO106 and at a sensitivity of 90%. The binary performance measures NPV, PPV, and specificity
were computed at this fixed sensitivity.
ROC analysis of EXO106 was used to further assess the discrimination between biopsy-positive
(for any cancer or for high-grade cancer) and biopsy–negative results and to compare EXO106
and SOC. Further, a logistic regression model combining EXO106 and SOC as predictors has
been added to the ROC analysis. Confidence intervals of AUCs were generated using nonparametric methods [2].
Correlation of the EXO106 Score with tumor grade was assessed by computing the pairwise
rank correlation by Spearman between the continuous EXO106 Score as one variable and the
three levels of cancer (i.e., no cancer, low-grade cancer [Gleason Grade 6] and high- grade
cancer [Gleason Grade ≥7]) as the second variable. A p-value was computed by Spearman’s rho
method.
Supplemental References:
[1] Brown LD, Cai TT, Das Gupta A. Confidence intervals for a binomial proportion and symptotic
expansions. The Annals of Statistics. 2002;30:160-201.
[2] DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas under two or more correlated
receiver operating characteristic curves: a nonparametric approach. Biometrics. 1988;44:837-45.