PMC3358358_01
Plots showing prostate-specific antigen (PSA) levels while undergoing hormonal treatment and cytotoxic and radiopharmaceutical treatment. Following a reduction in PSA while receiving leuprolide and bicalutamide at diagnosis (not shown), the patient's PSA began to rise. His PSA continued to rise despite anti-androgen withdrawal followed by ketoconazole, as shown. After discontinuing ketoconazole, his PSA spontaneously declined and did not rise for 9.8 months. Serum was drawn for enzyme-linked immunosorbent assay (ELISA) 3 times, in the intervals as shown, and an apheresis performed between the 6th and 7th doses of docetaxel. Durable episodes of freedom from progression in this context are unusual, and prompted a search for a mechanism linking his unusual course of metastatic melanoma with that of his metastatic prostate cancer. Another PSA decline was seen after treatment with docetaxel and 153Sm. The patient remained on leuprolide during the treatment course, and testosterone remained at castrate levels.
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F1
As shown in Figure 1, when his PSA reached 61, a 2-month trial of high-dose ketoconazole with hydrocortisone was started, again without an apparent favorable impact on his PSA.
Bone scan improvement 1 month, 3 months, and 8 months after discontinuing ketoconazole (left to right).
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F2
As shown in Figure 1, when his PSA reached 61, a 2-month trial of high-dose ketoconazole with hydrocortisone was started, again without an apparent favorable impact on his PSA. Bone scans performed three and eight months after ketoconazole was stopped also showed dramatic improvement in existing lesions (Figure 2).
Serum immunoglobulin and T cell responses to PSMA. 3a. Enzyme-linked immunosorbent assay (ELISA). Purified PSMA protein was used to coat EIA/RIA plates (Costar, Corning, NY), and after blocking with 5% non-fat dry milk, serum from the study patient drawn at month 23, 35, or 48, or from pooled normal human subjects (NHS), was diluted 1/100 in phosphate-buffered saline (PBS) and incubated overnight at 4 C. PBS served as an additional control. Plates were washed with PBS and a non-ionic detergent ( 0.1% Tween 20 [Sigma-Aldrich, St Louis, MO]) three times and then with PBS three times, and incubated with alkaline phosphatase (AP)-conjugated goat anti-human IgG (all isotypes) (Southern Biotech, Birmingham, AL, catalog #2040-04, 1:2000). Color development was accomplished by addition of AttoPhos reagents (Fisher Scientific, Pittsburgh, PA).. 3b,c. Intracellular cytokine assay (ICCA). PBMCs from an apheresis performed during month 54 were cultured for 10 days in RPMI 1640 + 10% fetal calf serum + IL-2 (10 IU/mL) and IL-15 10 (ng/mL) in the presence of one of four pools of 28-29 peptides from PSMA (pool #1). All cultures were performed in triplicate. After 10 days, cells in each well were divided into two cultures, one cultured with the original peptides and one without. After 1 hour, brefeldin (5muM) was added to all cultures to prevent cytokine release. Cells were cultured overnight and then stained for the surface markers CD3, CD4, CD8, and the intracellular marker INF-gamma and analyzed by flow cytometry on a BD LSR (Becton, Dickenson, Franklin Lakes, NJ). Cultures in which CD3+ CD4+ cells produced at least 2-fold more INF-gamma in the presence of peptide compared to cultures without peptide were considered positive and were reconfirmed. Cells were also stimulated with small peptide subpools until a single peptide was identified as the target.. 3c. Pools 16-20 and 3-28 are fully contained with the original pool#1, and both contained peptide 18, AHYDVLLSYPNKTHPNYISI (111-130). When this was retested, it was found to be the dominant epitope.
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F3
Patient sera were evaluated for antibodies specific to PSMA protein by ELISA (Figure 3). T cells (Figure 3). These are AHYDVLLSYPNKTHPNYISI (111-130); KVKMHIHSTNEVTRIYNV (340-358); YSLVHNLTKELKSPDEGF (471-488) and LVEKFYDPMFKYHLTVAQV (561-579).