molecular tests for the detection of prostate tumor derived nucleic acids in peripheral blood...
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Molecular tests for the detection of prostate tumor derived nucleic acids in peripheral bloodMatthias Jost, PhD1, John Day, PhD1, Ryan Slaughter1, Jack Groskopf, PhD1, Harry Rittenhouse, PhD1,
Theodore Koreckij, MD2, Deanna Gonzales2, Martin Kinnunen2, Bob Vessella, PhD2, and Mark Reynolds, PhD1
1Gen-Probe Incorporated, San Diego, CA; 2Department 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, PhDGen-Probe Incorporated
10210 Genetic Center DriveSan Diego, CA 92121
Phone: (858) [email protected]
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, 4x107 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: 7053-58.
(2) Groskopf et al. (2006) Clinical Chemistry 52:1089-95.
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
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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)
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phase contrast fluorescence
Figure 4 – PSA mRNA in CTC enriched fractions Figure 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 plasma Figure 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 CaptureTMA
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
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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 MeetingApril 18-22, 2009Denver, CO