H HR-MAS investigation of four potential markers for prostate cancer. 1 Mark G. SWANSON1, Daniel B. VIGNERON1, Joyce K. JAMES2, John KURHANEWICZ1 1University of California, San Francisco, 1 Irving Street, Box 1290, San Francisco, CA United States; 2Bruker Instruments, 47697 Westinghouse Drive, Fremont, CA United States; Introduction In previous three dimensional spectroscopic imaging (3D-MRSI) studies, changes in choline and citrate metabolism have been used to distinguish prostate cancer from healthy peripheral zone (PZ) tissues (1). The ability to metabolically detect prostate cancer could be further improved by identifying and combining additional markers. There is also a great need to correlate specific metabolic markers with cancer aggressiveness (grade) and their sensitivity to various therapies. Taurine, myo-inositol, and polyamine signals have recently been identified in the in situ prostate (2,3) and it has been suggested that myo-inositol is elevated in the presence of prostate cancer (4) while polyamines are reduced(3). Additionally, taurine changes have been observed in vitro, which may have diagnostic potential for prostate cancer (5). Since normal prostate tissue consists of a complex mixture of glandular and stromal components, which themselves have very different spectroscopic features, higher resolution ex vivo studies are necessary to better understand metabolic changes observed in vivo. Previous high resolution magic angle spinning (1H HR-MAS) NMR studies have shown that choline, phosphocholine, and glycerophosphcholine, are the major compounds responsible for the in vivo elevation of choline in prostate cancer(6). The purpose of the present study was to investigate four potential metabolic markers for prostate cancer, taurine, myo- and scyllo-inositol, and polyamines, using 1H HR-MAS spectroscopy of post surgical prostate tissue. Methods Twenty seven patients with biopsy proven prostate cancer underwent an endorectal MRI/3D-MRSI staging examination prior to radical prostatectomy . Upon prostate removal, MRI/3D-MRSI reports were used to target tissue samples (n=54) from the peripheral zone. Samples were stored in liquid nitrogen until 1H HR-MAS analysis. 1H HR-MAS spectra were acquired on a 9.4 T Bruker Avance NMR spectrometer. Samples were spun at 2.5 kHz using a 4 mm 1H/13C HR-MAS probe. 1D parameters: 20 °C; 16K points; 8kHz spectral width; zgpr pulse sequence with HOD presaturation. For selected samples, 2D J-Resolved and TOCSY experiments (mixing time = 65 ms) were also performed. After 1H HR-MAS spectroscopy, samples were weighed (mean: 7.68± 1.79 mg), transferred to cassettes, and submitted in formalin for histology. During pathologic analysis, samples were imbedded in paraffin wax and 3 level sections were reviewed for each. Standard hemotoxylin and eosin staining was used. Sample compositions were reported in terms of percent cancer, benign tissue, glandular components, stroma, prostatitis, fibrosis, and atrophy. 1H HR-MAS data were analyzed off-line using MacNUTS (Acorn NMR). Data were apodized with a 0.3 Hz exponential function prior to FT. The creatine resonance at 3.04 ppm was used as an internal chemical shift reference. Resonances were assigned from previously reported chemical shift values, comparison to standards, and analysis of 2D crosspeaks. Peak areas were determined by Lorentzian and Gaussian curve-fitting following baseline correction of the region of interest. Metabolite ratios were compared using a two-tailed student's t-test, assuming different sample sizes and variances. Results As shown in Figure 1, mean taurine/creatine, myo-inositol/creatine, and scyllo-inositol/creatine ratios were higher in prostate cancer than in healthy glandular tissue; however, the only significant difference was for taurine/creatine (p=0.03). Mean taurine/creatine, myoinositol/creatine, and scyllo-inositol/creatine ratios for healthy stromal tissues were also not significantly different than for healthy glandular tissue or prostate cancer. Polyamine/creatine ratios were significantly higher in healthy glandular tissues than in stromal tissues (p=0.04) and prostate cancer (p=0.0003). As anticipated, polyamine/creatine ratios between healthy stromal tissues and prostate cancer were not significantly different due to a reduction in polyamines in both tissues. As shown in Figure 2, myo-inositol/creatine and scylloinositol/creatine ratios were higher for the more aggressive Gleason 8+9 cancers than for less aggressive Gleason 6+7 cancers. Polyamine/creatine ratios also decreased with prostate cancer grade, suggesting a further reduction of polyamines with cancer aggressiveness. However, because of the small number of samples in each cancer group, the differences in myo-inositol/creatine, scylloinositol/creatine, and polyamine/creatine ratios did not reach statistical significance in this study. Figure 1. Mean taurine/creatine, myo-inositol/creatine, scylloinositol/creatine, and polyamine/creatine ratios in regions of healthy glandular tissue (n=29), stromal tissue (n=12), and prostate cancer (n=13). Figure 2. Mean taurine/creatine, myo-inositol/creatine, scylloinositol/creatine, and polyamine/creatine ratios for Gleason 6 (n=6), Gleason 7 (n=3), and Gleason 8+9 (n=4) grade prostate cancers. Discussion The most important findings of this study were that taurine/creatine increased significantly while polyamine/creatine levels decreased significantly for prostate cancer vs. healthy glandular tissues. Although myo- and scyllo-inositol/creatine levels were also higher in prostate cancer vs. healthy glandular tissues, the differences were not statistically significant. Myo- and scyllo-inositol/creatine levels were also higher in the most aggressive cancers, suggesting that these metabolites should be further investigated in a larger cohort of patients. Finally, polyamine/creatine levels decreased with increasing cancer grade, suggesting that polyamine reduction could serve as an additional marker for prostate cancer presence and aggressiveness. These studies suggest that taurine and polyamines could potentially be used as in vivo markers for prostate cancer, while myo- and scylloinositol should be further studied before any conclusions can be drawn. References 1. 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