1. Rapid Next-Generation Sequencing Method for Prediction of Prostate Cancer Risks 
Viacheslav Y. Fofanov, Kinnari Upadhyay, Alexander Pearlman, Johnny Loke, Vivian O, Yongzhao Shao, Stephen Freedland, Harry Ostrer 
The Journal of Molecular Diagnostics 
Volume 21, Issue 1, Pages (January 2019)
DOI: /j.jmoldx
Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Schematic of copy number alteration (CNA) target probe set and anchor probe set placement. The normalized (standard normal distribution) CNA log-rank scores for several cohorts of prostate cancer research samples are shown (as originally described5). Log-rank scores outside 2 SDs from the mean (here, >2 or <−2) indicate statistically significant evidence of a copy number gain or loss, respectively. CNA target probe sets [red rectangles, not to scale, with left-hand side (LHS) and right-hand side (RHS)] are placed throughout the region known to exhibit CNAs (here, around the CC2D1A gene). Anchor probe sets (green rectangles, not to scale, with LHS and RHS) are placed in regions previously identified as copy number invariant. The number of sequenced amplification products for CNA target probe sets, normalized by the number of sequenced amplification products for anchor probe sets, serves to estimate copy number (copy number gain in the example shown). MB, megabase.
The Journal of Molecular Diagnostics  , 49-57DOI: ( /j.jmoldx )
Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Log-rank scores for prostate cancer surgical research sample. Probe sets are in order of their physical location along the human genome (ie, probes on chromosome 1 appear before those on chromosome 2). Because all copy number alteration (CNA) target probe sets are normalized by number of reads from normal (noncancer) samples, probe sets with normalized log-rank scores that fall outside predetermined thresholds (black) indicate potential CNA gain or loss events, whereas those near 0 (gray) indicate CNA neutral events. For regions of interest (ROIs) that contain more than one probe set (most in our assay), gain or loss determinations are made by averaging normalized log-rank scores for all probe sets in the ROI before comparison with predetermined thresholds.
The Journal of Molecular Diagnostics  , 49-57DOI: ( /j.jmoldx )
Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Copy number alteration (CNA) panel optimization. Performance characteristics of each of the 1222 probe sets in the CNA panel were evaluated in the context of the whole panel, using a test set of 96 healthy (noncancer) and cancer cell lines. Probe sets that consistently produced a low number (eg, probe 1603 in A, COL19A1 gene) or high number (eg, probe 804 in B, CDH2 gene) of reads were defined as falling outside the range requirements (red). Similarly, probe sets with consistently high variance in number of reads across technical replicates (eg, probe 804 in B) were defined as falling outside technical reproducibility requirements (red). These probe sets physically remained in the panel but were discarded from subsequent bioinformatics analyses.
The Journal of Molecular Diagnostics  , 49-57DOI: ( /j.jmoldx )
Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Reproducibility and robustness of assay. Metastasis potential scores (MPSs) were computed based on the next-generation copy number alteration assay using 50 ng of starting material from 87 prostate cancer surgical research samples. Linear trendline (dotted lines), associated linear regression value, and Pearson correlation coefficient (Pearson Corr) values are provided. A: Technical replicates for all 87 samples highly positively correlate. B: The MPSs based on the assay are resistant to loss of the probes with low reads.
The Journal of Molecular Diagnostics  , 49-57DOI: ( /j.jmoldx )
Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Figure 5
Effect of sample input amount on assay reproducibility. Metastasis potential scores (MPSs) were computed based on the next-generation copy number alteration assay from reactions that had 25-, 12-, and 5-ng inputs. Linear trendline (dotted lines), associated linear regression values, and Pearson correlation coefficient (Pearson Corr) values are provided. There is a high positive agreement between 25- and 12-ng input (A); there is less agreement between 5- and 25-ng input (B).
The Journal of Molecular Diagnostics  , 49-57DOI: ( /j.jmoldx )
Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7. Figure 6
Comparisons to microarray-derived metastasis potential scores (MPSs) and applicability to punched biopsy samples. A: A total of 70 prostate cancer surgical research (PCSR) samples with a wide range of previously established metastatic potentials were evaluated using the next-generation copy number alteration (NG-CNA) panel and OncoScan CNA assay approaches.5,6 Linear trendline (dotted lines), associated linear regression values, and Pearson correlation coefficient (Pearson Corr) values are provided. Reproducibility between the two approaches is high. B: Eight PCSR samples (punched biopsy–matched samples) were evaluated with the NG-CNA panel, with highly reproducible results.
The Journal of Molecular Diagnostics  , 49-57DOI: ( /j.jmoldx )
Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

8. Supplemental Figure S1
Comparison of OncoScan arrayCGH assay–derived metastasis potential scores (MPSs) for indolent primary tumor (iPT) and metastasis-prone primary tumor (mPT) samples. Statistically significant differences (P = 0.002) between MPSs have been observed for iPT and mPT samples.
The Journal of Molecular Diagnostics  , 49-57DOI: ( /j.jmoldx )
Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions