Molecular tests for the detection of prostate tumor derived nucleic acids in peripheral blood Matthias Jost, PhD 1, John Day, PhD 1, Ryan Slaughter 1, Jack Groskopf, PhD 1, Harry Rittenhouse, PhD 1, Theodore Koreckij, MD 2, Deanna Gonzales 2, Martin Kinnunen 2, Bob Vessella, PhD 2, and Mark Reynolds, PhD 1 1 Gen-Probe Incorporated, San Diego, CA; 2 Department of Urology, University of Washington, Seattle, WA Background Prostate cancer is the second leading cause of cancer mortality in American men. Although the serum PSA test is widely used for early detection, more specific prognostic tests are needed to guide treatment decisions. Recently, the enumeration of circulating tumor cells (CTCs) has been shown to correlate with disease recurrence and metastasis following definitive treatment (1). We hypothesized that processing specimens using immunomagnetic CTC enrichment methods prior to testing could enhance the sensitivity and specificity of amplified molecular tests for prostate-derived nucleic acids. Here we report detection of PSA, PCA3, and TMPRSS2:ERG gene fusion mRNAs in peripheral blood specimens processed by an immunomagnetic fractionation procedure. Methods Blood from 5 benign prostatic hyperplasia (BPH) patients and 62 prostate cancer patients (including 33 advanced-stage and 29 early- stage patients) was collected into EDTA-containing Vacutainer® tubes under IRB-approved protocols. Whole blood (5 mL) was processed to enrich CTCs with antibody-coated magnetic particles (anti-PSMA and/or anti-EpCAM). PSA, PCA3, and TMPRSS2:ERG gene fusion mRNAs were isolated, amplified, and detected with prototype assays that utilize Gen-Probe's magnetic Target Capture, Transcription-Mediated Amplification, and Hybridization Protection Assay technologies. These mRNAs were also isolated, amplified, and detected using these prototype assays in matched blood and plasma samples. Results Our immunomagnetic CTC enrichment method provided robust capture and detection of as few as 5 cells per 5 mL of blood in model system experiments. Applying this CTC enrichment method, specimens from 34% of the advanced-stage prostate cancer patients had positive results for 1 or more of our amplified molecular assays, whereas similarly processed specimens from BPH patients had negative results, as expected. Interestingly, for those advanced-stage patients whose CTC- enriched specimens had positive results, almost all of the matched plasma samples also had positive results, although with lower amplified mRNA signals compared to the corresponding CTC-enriched fractions. Outlook Additional refinements are underway to increase the sensitivity of detection in the pre-radical prostatectomy (pre-RP) population. Abstract (updated) Materials and Methods Clinical Specimen DataCell Capture Model System Data Matthias Jost, PhD Gen-Probe Incorporated Genetic Center Drive San Diego, CA Phone: (858) The molecular assays described herein have not been approved or cleared by the FDA. Magnetic capture of epithelial cells from peripheral blood Magnetic particles (Dynal, 4.5 m diameter, 4x10 7 particles per reaction) were coated with anti-EpCAM (Dynal) and anti-PSMA antibodies and incubated with up to 5 mL EDTA blood for 30 min at room temperature. Magnetic-bound fractions were subjected to three washing steps with PBS containing 0.2% BSA and subsequently treated with Gen-Probe lysis/stabilization buffer. Specimen processing up to this point was carried out at University of Washington; molecular testing was done at Gen-Probe. Cell lines C4-2, LNCaP and GFP-LNCaP cells (kindly provided by Dr. Srivastava, Center for Prostate Disease Research) were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum under standard culture conditions. In addition, GFP- LNCaP medium contained G418 (0.5 mg/mL) and C4-2 medium contained L-glutamine (2 mM). Cell spiking of individual cells was done with an Eppendorf micro-manipulator, or by manual pipetting under an inverted Olympus CK2 microscope. Micrographs were taken with a Zeiss Axio Imager Z.1 microscope. Whole blood lysis and plasma sample collection Whole blood was collected in EDTA Vacutainers. Reproducible cell capture and molecular detection was demonstrated with spiked prostate cells. CTC-enriched fractions from advanced-stage cancers contained prostate-specific mRNAs. Not found in BPH and early-stage prostate cancers. PSA mRNA exhibited highest sensitivity. PSA mRNA was also detected in plasma of advanced-stage cancers. The TMPRSS2:ERG gene fusion was detected in a subset of the advanced-stage cancers. Anti-EpCAM coated magnetic particles exhibited slightly higher sensitivity as compared to anti- EpCAM plus anti-PSMA coated magnetic particles. References (1) Danila et al. (2007) Clinical Cancer Research 13: (2) Groskopf et al. (2006) Clinical Chemistry 52: Subsequently, 0.4 mL of whole blood and 2 mL of matched plasma were treated with 2 mL of Gen-Probe lysis/stabilization buffer. Molecular analysis of prostate specific mRNA All specimens were kept at –70ºC until further processing. Target mRNA was captured directly out of specimens with specific oligonucleotides conjugated to magnetic particles (Target Capture) and subjected to nucleic acid testing with an amplified nucleic acid test based on Transcription-Mediated Amplification (TMA) (2). Acknowledgements We would like to thank the patients for their participation in this study. In addition, we would like to thank Drs. Lange, Ellis and Montgomery for their aid in patient accrual. Figure 1 A-C. Micrograph series Conclusions 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 5 cells10 cells25 cells PSA mRNA copies Figure 2 – Magnetic capture of spiked LNCaP cells Figure 3 – Magnetic capture of spiked C4-2 cells GFP-LNCaP cells were spiked into EDTA blood (1A) and incubated with anti-EpCAM coated magnetic particles. The particle-bound fraction displayed GFP-LNCaP cells (1B) and a low amount of putative, non-fluorescent blood cells (1C). Reproducibility of LNCaP cell capture with anti-EpCAM magnetic particles out of whole blood (8 cells spiked into 4 mL EDTA blood). Magnetic capture of C4-2 cells with anti-EpCAM magnetic particles out of 5 mL EDTA blood. N=10 (5, 10 cells), n=5 (25 cells) 1 A 1 B 1 C phase contrastfluorescence Figure 4 – PSA mRNA in CTC enriched fractionsFigure 7 – PCA3 mRNA in CTC enriched fractions Magnetic particle combo (anti-EpCAM + anti-PSMA coated particles) enriched fractions. PSA mRNA was detected in 9/16 androgen-independent and 2/17 androgen-dependent samples. Each bar represents one patient specimen. Magnetic particle combo enriched fractions. PCA3 mRNA was detected in 5/16 androgen-independent and 0/17 androgen- dependent samples. Copy numbers are lower as compared to PSA mRNA. Figure 5 – PSA mRNA in plasmaFigure 8 – TMPRSS2:ERG mRNA in CTC enriched fractions PSA mRNA detection directly in plasma fractions. The result highly correlated with CTC enriched fractions. Positive detection of 8/16 androgen-dependent and 2/17 androgen-dependent specimens. Gaps indicate data not available. Magnetic particle combo enriched fractions. TMPRSS2:ERG mRNA was detected in 3/14 androgen-independent and 1/14 androgen-dependent samples. Gaps indicate data not available. Figure 6 – PSA mRNA in whole blood PSA mRNA detection in whole blood fractions. Gaps indicate data not available. Target Capture TMA Target Capture Small magnetic particles with synthetic oligonucleotides bound to the surface are used to capture the target nucleic acid directly out of many different kinds of body fluids, e.g. plasma, blood, saliva, and urine. The magnetic particles are subsequently drawn to the side of the test tube by a magnet, and wash steps remove substances that can potentially interfere with the TMA assay. TMA is an RNA transcription amplification system using two enzymes to drive the reaction: RNA polymerase and reverse transcriptase. Workflow PSA mRNA was detected in 7/13 anti-EpCAM enriched fractions and 6/13 fractions enriched with the particle combo. Figure 9 – CTC enrichment with anti-EpCAM versus anti- EpCAM plus anti-PSMA magnetic particles 2009 AACR Annual Meeting April 18-22, 2009 Denver, CO