High-Resolution Genomic Profiling of Disseminated Tumor Cells in Prostate Cancer  Yu Wu, Jamie R. Schoenborn, Colm Morrissey, Jing Xia, Sandy Larson, Lisha G. Brown, Xiaoyu Qu, Paul H. Lange, Peter S. Nelson, Robert L. Vessella, Min Fang  The Journal of Molecular Diagnostics  Volume 18, Issue 1, Pages 131-143 (January 2016) DOI: 10.1016/j.jmoldx.2015.08.004 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 WGA and SNP array profiling on LNCaP populations of one and five- cells are reproducible and accurate. A: Representative images of LNCaP from bulk gDNA (DNA extracted from 2 × 106 cells), WGA2 (GenomePlex Complete Whole Genome Amplification kit)-amplified from 10 ng DNA, and Picoplex WGA amplified five- and one-cell aliquots of LNCaP. B: Schematic presentation of chromosome 10 (82,394,009 to 106,849,461 bp, red box) showing three deletions of 4.46 Mb, 0.71 Mb, and 1.94 Mb in size. The Hudson alpha track from University of California Santa Cruz genome browser was aligned to the corresponding regions. SNP, single nucleotide polymorphism; WGA, whole-genome amplification. The Journal of Molecular Diagnostics 2016 18, 131-143DOI: (10.1016/j.jmoldx.2015.08.004) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 SNP array is able to detect SCNAs when present in ≥50% of the population. A: Results from 5-cell mixtures of a 22q-deletion syndrome (VCF) cell line and NFR cells. The VCF cells, characterized by a 2.79-Mb deletion on 22q, were combined with female GM10473 lymphoblasts. The 5VCF, 4VCF:1NFR, and 3VCF:2NFR mixtures all demonstrated visible deletion of 22q (shaded in pink), whereas no deletion was detected in the 2VCF:3NFR and 1VCF:4NFR mixtures. B: The size, MPI (representing the magnitude of deletion), and P value (significance) of deletion calls of the cell mixtures. C: Results from 20-cell mixtures of LNCaP cells with normal female GM10966 lymphoblasts. The SCNAs in bulk LNCaP DNA were seen in the combination of 15LNCaP:5NFR and 10LNCaP:10NFR mixtures but not in the 5LNCaP:15NFR mixture. MPI, mean probe intensity; NFR, normal female lymphoblast reference; SCNA, somatic copy number aberration; SNP, single nucleotide polymorphism; VCF, velocardiofacial The Journal of Molecular Diagnostics 2016 18, 131-143DOI: (10.1016/j.jmoldx.2015.08.004) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Genomic profiles of DTC from patients with advanced prostate cancer show highly aberrant genomes. A: Combined genome profile of the eight Adv-DTCs from eight different patients, with selected genes of interest highlighted. B: Individual genomic profile results for the eight patients. The x axis indicates the patient case number or cell line. y axis indicates the percentages of genome of each sample that is normal (gray), CN gain/amplification (blue), and CN loss (red). The cell numbers in the samples were 20 cells for patient 2451, 20 cells for patient 2380C1, 10 cells for patient 2373, 10 cells for patient 2299, 13 cells for patient 2439, 5 cells for patient 2693, 10 cells for patient 2474, and 20 cells for patient 2346. The bulk LNCaP DNA result is shown on the far right. C: The distribution of size and type of CNAs in DTCs relative to normal and tumor samples. Dark bars indicate CN variations that are common among the population, whereas yellow and light blue indicate CNAs that are considered private and likely to be tumor specific. Normals include CD45+ lymphocytes from the blood or bone marrow. D: A representative DTC profiling (2380C1) is shown. Adv, advanced; CN, copy number; CNA, copy number aberration; DTC, disseminated tumor cell. The Journal of Molecular Diagnostics 2016 18, 131-143DOI: (10.1016/j.jmoldx.2015.08.004) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Validation of CN alterations in Adv-DTC. A: Representative Nanostring nCounter results for individuals 2380, 2474, and 2299. Red dashed line represents CN = 2 (normal) values in Illumina SNP-CGH, red faded box represents Nanostring CN = 1.5–2.5 normal range. B: Overall validation of CNAs detected in six individuals with the use of Nanostring nCounter assay (Loss: true = CN ≤ 1.4, Ambiguous = CN1.5–1.6, false = CN ≥ 1.7; Gain: true = CN ≥ 2.6, ambiguous = CN2.4–2.5, false = CN ≤ 2.3). C: Quantitation of AR amplification in 2474; other DTC and CD45+ samples do not have AR amplification. D: Quantitation of PTEN loss in 2299 and 2439; other DTC and CD45+ samples have normal PTEN CN. Dashed gray lines in B and C represent mean CN of nonaberrant samples. Adv, advanced; CGH, comparative genome hybridization; CN, copy number; CNA, copy number aberration; DTC, disseminated tumor cell; SNP, single-nuclear polymorphism. The Journal of Molecular Diagnostics 2016 18, 131-143DOI: (10.1016/j.jmoldx.2015.08.004) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Comparison of matched DTC and metastatic tumors from two individuals supports their relation. A: SNP array whole genome plot of DTC 2299 and three patient-matched bone mets; blue represents CN gains and red represents loss. B: SNP array data of DTC 2439 and three patient-matched soft tissue mets. Yellow horizontal bars in samples denote regions ≥3 Mb that include copy-neutral LOH . C: Comparison of the percentage of each genome affected by SCNAs and CN LOH (note, CN LOH could not be determined for DTCs). D: Percentage of SCNAs in DTC 2299 and DTC 2439 that are also detected in the matched metastatic tumors. CN, copy number; DTC, disseminated tumor cell; LN, lymph node; LOH, loss of heterozygosity; mets, metastasis; SCNA, somatic copy number aberrations; SNP, single-nuclear polymorphism. The Journal of Molecular Diagnostics 2016 18, 131-143DOI: (10.1016/j.jmoldx.2015.08.004) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions