Method of diagnosing monitoring, staging, imaging and treating prostate cancer

The present invention provides new markers and methods for detecting, diagnosing, monitoring, staging, prognosticating, imaging and treating prostate cancer.

EXAMPLES The present invention is further described by the following example. The example is provided solely to illustrate the invention by reference to specific embodiments. This exemplification, while illustrating certain aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention. All examples outlined here were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniques of the following example can be carried out as described in standard laboratory manuals, such as Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Relative Quantitation of Gene Expression Real-Time quantitative PCR with fluorescent Taqman probes is a quantitation detection system utilizing the 5′-3′ nuclease activity of Taq DNA polymerase. The method uses an internal fluorescent oligonucleotide probe (Taqman) labeled with a 5′ reporter dye and a downstream, 3′ quencher dye. During PCR, the 5′-3′ nuclease activity of Taq DNA polymerase releases the reporter, whose fluorescence can then be detected by the laser detector of the Model 7700 Sequence Detection System (PE Applied Biosystems, Foster City, Calif., USA). Amplification of an endogenous control is used to standardize the amount of sample RNA added to the reaction and normalize for Reverse Transcriptase (RT) efficiency. Either cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ATPase, or 18S ribosomal RNA (rRNA) is used as this endogenous control. To calculate relative quantitation between all the samples studied, the target RNA levels for one sample were used as the basis for comparative results (calibrator). Quantitation relative to the “calibrator” can be obtained using the standard curve method or the comparative method (User Bulletin &num;2: ABI PRISM 7700 Sequence Detection System). The tissue distribution and the level of the target gene were evaluated for every sample in normal and cancer tissues. Total RNA was extracted from normal tissues, cancer tissues, and from cancers and the corresponding matched adjacent tissues. Subsequently, first strand cDNA was prepared with reverse transcriptase and the polymerase chain reaction was done using primers and Taqman probes specific to each target gene. The results were analyzed using the ABI PRISM 7700 Sequence Detector. The absolute numbers are relative levels of expression of the target gene in a particular tissue compared to the calibrator tissue. Expression of Clone ID 824430 (Pro119) For the CSG Pro119, real-time quantitative PCR was performed using the following primers: Forward Primer 5′-GGGCTTGTCACAGTCTCTACTGTT-3′ (SEQ ID NO:8) Reverse Primer 5′-GCCAGAACATTGTGAGCACAC-3′ (SEQ ID NO:9) The absolute numbers depicted in Table 1 are relative levels of expression of the CSG referred to as Pro119 in 12 normal different tissues. All the values are compared to normal prostate (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals. 1 TABLE 1 Relative Levels of CSG Pro119 Expression in Pooled Samples Tissue NORMAL Brain 0.002 Heart 0.001 Kidney 0.001 Liver 0.003 Lung 0.006 Mammary 0.003 Muscle 0.003 Prostate 1 Small Intestine 0.006 Testis 0.009 Thymus 0.003 Uterus 0.02 The relative levels of expression in Table 1 show that Pro119 mRNA expression is higher in prostate (1.0) compared with all other normal tissues analyzed. All other tissues analyzed show a relative Pro119 mRNA expression of less than one. The absolute numbers in Table 1 were obtained analyzing pools of samples of a particular tissue from different individuals. They cannot be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 2. The absolute numbers depicted in Table 2 are relative levels of expression of Pro119 in 49 pairs of matched and 15 unmatched samples. All the values are compared to normal prostate (calibrator). A matching pair is formed by obtaining mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual. 2 TABLE 2 Relative Levels of CSG Pro119 Expression in Individual Samples Matching Normal Sample ID Tissue Cancer Adjacent Pro12B Prostate 1 0.13 0.16 ProC234 Prostate 2 1.73 Pro78XB Prostate 3 15.9 2.9 Pro109XB Prostate 4 0.13 0.21 Pro84XB Prostate 5 25.79 0.49 Pro101XB Prostate 6 20.33 9.35 Pro91X Prostate 7 19.26 0.85 Pro13XB Prostate 8 0.07 0.16 ProC215 Prostate 9 4.52 Pro125XB Prostate 10 0.09 0.05 Pro23B Prostate 11 0.86 1.05 Pro90XB Prostate 12 0.01 0.23 ProC280 Prostate 13 0.28 Pro20XB Prostate 14 2.68 0 Pro34B Prostate 15 2.96 1.84 Pro110 Prostate 16 0.19 0.5 Pro65XB Prostate 17 16.02 0.01 Pro69XB Prostate 18 2.26 1.02 Pro326 Prostate 19 0.81 0.72 Pro10R Prostate 20 1.68 (Prostatitis) Pro20R Prostate 21 0.71 (Prostatitis) ProC258 Prostate 22 (BPH) 0.03 Pro263C Prostate 23 (BPH) 4.37 Pro267A Prostate 24 (BPH) 0.02 Pro27lA Prostate 25 (BPH) 0.49 Pro460Z Prostate 26 (BPH) 0.4 ProC032 Prostate 27 (BPH) 0.97 Bld32XK Bladder 1 0 0.01 Bld46XK Bladder 2 0.01 0.01 Bld66X Bladder 3 0.02 0 Kid106XD Kidney 1 0.01 0 Kid107XD Kidney 2 0 0 Kid109XD Kidney 3 0.02 0 Tst239X/240X Testis 1 0.004 0.004 Tst Testis 2 0.03 0.01 S9820647A/B Tst Testis 3 0 0 S9820663A/B Skn248S Skin 1 0.03 0 Skn287S Skin 2 0 0 SmI9807A212A/ Small Intestine 1 0.01 0.004 213A SmI9802H008/ Small Intestine 2 0.003 0.001 H009 ClnAC19 Colon 1 0.02 0.62 ClnAS12 Colon 2 0.01 0.32 ClnAS43 Colon 3 0.02 0.15 Sto115S Stomach 1 0 0.02 Sto15S Stomach 2 0.01 0 Sto17S Stomach 3 0.01 0.01 Lng476Q/477Q Lung 1 0 0 Lng60XL Lung 2 0 0 Lng75XC Lung 3 0 0 Pan921O/922O Pancreas 1 0.01 0.02 Pan9408C044R/ Pancreas 2 0 0 45R Pan82XP Pancreas 3 0.01 0 Utr1359O/ Uterus 1 0.05 0 1358O Ultr1417O/ Uterus 2 0 0.02 1418O Utr233U96/ Uterus 3 0.03 0.01 234U96 Utr850U/851U Uterus 4 0.01 0.03 End10479B/ Endometrium 1 0.04 0.13 10479D End9705A125A/ Endometrium 2 0 0.02 126A Mam14l53a1c/ Mammary Gland 1 0 0 a2f Mam00014D05/ Mammary Gland 2 0.1 0 N05 Mam162X Mammary Gland 3 0 0 Mam19DN Mammary Gland 4 0 0 Ovr1005O Ovary 1 0.001 0vr1028O Ovary 2 0.03 Ovr1037O/ Ovary 3 0.02 0.004 1038O Ovr9702C018G Ovary 4 0.03 Ovr206I Ovary 5 0.02 0 &equals; Negative In the analysis of matching samples, the higher levels of expression were in prostate, showing a high degree of tissue specificity for prostate tissue. Eleven of 16 prostate matching samples showed upregulation in Pro119 mRNA expression. Of all the samples different than prostate analyzed, no other cancer or normal tissue sample showed expression levels comparable to the mRNA expression in prostate. These results confirm a high degree of tissue specificity as obtained with the panel of normal pooled samples (Table 1). Furthermore, the level of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual were compared. This comparison provides an indication of specificity for the cancer (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Table 2 shows overexpression of Pro119 in 11 out of 16 primary prostate cancer tissues compared with their respective normal adjacent. Thus, there was overexpression in the cancer tissue for 68.75% of the prostate matching samples tested. Altogether, the high degree of tissue specificity, plus the mRNA overexpression in 68.75% of the prostate matching samples tested are indicative of Pro119 being a diagnostic marker for prostate cancer. Expression of Clone ID788274 (Pro121): For the CSG Pro121, real-time quantitative PCR was performed using the following primers: Forward Primer 5′-CGCCCATTTCTCAGATCAAG-3′ (SEQ ID NO:10) Reverse Primer 5′-CGCCCAGTAGATGTTTCAAAG-3′ (SEQ ID NO:11) The absolute numbers depicted in Table 3 are relative levels of expression of the CSG Pro121 in 12 normal different tissues. All the values are compared to normal testis (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals. 3 TABLE 3 Relative Levels of CSG Pro121 Expression in Pooled Samples Tissue NORMAL Brain 0 Heart 0 Kidney 0 Liver 0 Lung 0 Mammary 0 Muscle 0.2 Prostate 1612.4 Small Intestines 0.14 Testis 1 Thymus 0 Uterus 1.5 The relative levels of expression in Table 3 show that Pro121mRNA expression is significantly higher (1612.4) in prostate compared with all other normal tissues analyzed. All other tissues analyzed show a relative Pro121 mRNA expression of zero except uterus (1.5), testis (1.0), muscle (0.2) and small intestine (0.14). The absolute numbers in Table 3 were obtained analyzing pools of samples of a particular tissue from different individuals. They cannot be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 4. The absolute numbers depicted in Table 4 are relative levels of expression of Pro121 in 50 pairs of matched and 16 unmatched samples. All the values are compared to normal testis (calibrator). A matching pair is formed by obtaining mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual. 4 TABLE 4 Relative Levels of CSG Pro121 Expression in Individual Samples Matching Normal Sample ID Tissue Cancer Adjacent Pro12B Prostate 1 15883.4 882.21 ProC234 Prostate 2 2511.6 Pro78XB Prostate 3 2872.6 4651 Pro109XB Prostate 4 311.71 219.03 Pro84XB Prostate 5 14512.7 4907.8 Pro101XB Prostate 6 24876.1 14314.9 Pro91X Prostate 7 7553.7 1623.6 Pro13XB Prostate 8 84.93 31.57 ProC215 Prostate 9 12917.2 Pro125XB Prostate 10 50.39 294.49 Pro23B Prostate 11 8085.9 3571.47 Pro90XB Prostate 12 16670.2 4597 ProC280 Prostate 13 13219.8 Pro20XB Prostate 14 2782.6 0 Pro34B Prostate 15 14913.87 8575.76 Pro110 Prostate 16 4046.55 5231.36 Pro65XB Prostate 17 3064.75 7879.12 Pro69XB Prostate 18 222.4 151.7 Pro326 Prostate 19 14179.84 4748.68 Pro10R Prostate 20 4025.72 (Prostatitis) Pro20R Prostate 21 3917.86 (Prostatitis) ProC258 Prostate 22 (BPH) 1083.11 Pro263C Prostate 23 (BPH) 3436.64 Pro267A Prostate 24 (BPH) 1295.86 Pro27lA Prostate 25 (BPH) 614 Pro460Z Prostate 26 (BPH) 2682.9 ProC032 Prostate 27 (BPH) 489.48 Bld32XK Bladder 1 2.55 31.64 Bld46XK Bladder 2 5.58 7.52 Bld66X Bladder 3 1823.8 0 Kid106XD Kidney 1 0 0 Kid107XD Kidney 2 0 0 Kid109XD Kidney 3 0 0 Tst239X/ Testis 1 34.27 0 240X TstS9820647A/ Testis 2 0 0 B TstS9820663 Testis 3 189.7 0 Sto115S Stomach 1 0 0 Sto15S Stomach 2 37.79 0 Sto17S Stomach 3 78.42 0 SmI9807A212A/ Small Intestine 1 0.88 0 213A SmI9802H008/ Small Intestine 2 2.04 0 H009 C1nAC19 Colon 1 377.3 0 ClnAS12 Colon 2 1308.8 0 ClnAS43 Colon 3 0 0 Lng476Q/477Q Lung 1 0 0 Lng60XL Lung 2 0 0 Lng75XC Lung 3 0 0 Pan921O/922O Pancreas 1 0 0 Pan9408C044R/ Pancreas 2 0 0 45R Pan82XP Pancreas 3 1.03 0 Skn248S Skin 1 0 0 Skn287S Skin 2 0 0 Utr1359O/ Uterus 1 0 0 1358O Utr1417O/ Uterus 2 0 0 1418O Utr233U96/ Uterus 3 4.5 0 234U96 Utr850U/851U Uterus 4 0 0 End10479B/ Endometrium 1 89.13 0 10479D End9705A125A/ Endometrium 2 0 0 126A Mam14153alc/ Mammary Gland 1 0 0 a2f Mam00014D05/ Mammary Gland 2 0 0 N05 Mam162X Mammary Gland 3 0 1.28 Mam14DN Mammary Gland 4 0 0 Mam19DN Mammary Gland 5 0 0 Ovr1005O Ovary 1 2.68 Ovr1028O Ovary 2 0 Ovr1037O/ Ovary 3 0 0 1038O Ovr9702C018G Ovary 4 0 Ovr206I Ovary 5 0 In the analysis of matching samples, the higher levels of expression were in prostate, showing a high degree of tissue specificity for prostate tissue. Of all the samples different than prostate analyzed, only 2 cancer samples (the cancer sample bladder 3 with 1823.8 and colon 2 with 1308.8 showed an expression comparable to the mRNA expression in prostate. Other cancer tissues with some expression were: cancer samples colon 1 (377.3), testis 3 (189.7), stomach 3 (78.42), stomach 2 (37.79), testis 1 (34.27), bladder 2 (5.58) and normal adjacent tissue samples bladder 1 (31.64) and bladder 2 (7.52). All remaining tissue samples had relative Pro121 mRNA expression levels of less than 5. These results confirmed a high degree of tissue specificity as obtained with the panel of normal pooled samples (Table 3). Furthermore, the level of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual were compared. This comparison provides an indication of specificity for the cancer (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Table 4 shows overexpression of Pro121 in 12 out of 16 primary prostate cancer tissues compared with their respective normal adjacent. Thus, there was overexpression in the cancer tissue for 75% of the prostate matching samples tested. Altogether, a high tissue specificity, plus the mRNA overexpression in 75% of the prostate matching samples tested are indicative of Pro121 being a diagnostic marker for prostate cancer. Expression of Clone ID 832357 (Pro124) For the CSG Pro124, real-time quantitative PCR was performed using the following primers: Forward Primer 5′-AAGGGAATGGTATAGAATTGGAGAG-3′ (SEQ ID NO:12) Reverse Primer 5′-CCTGCTCAAATACCACCACTTC-3′ (SEQ ID NO:13) The absolute numbers depicted in Table 5 are relative levels of expression of the CSG Pro124 in 12 normal different tissues. All the values are compared to normal testis (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals. 5 TABLE 5 Relative Levels of CSG Pro124 Expression in Pooled Samples Tissue NORMAL Brain 0.26 Heart 0 Kidney 0 Liver 9.19 Lung 0 Mammary 0.05 Muscle 0 Prostate 117.38 Small Intestine 0 Testis 1 Thymus 0.4 Uterus 0.92 The relative levels of expression in Table 5 show that Pro124 mRNA expression is very high in normal prostate (117.38) compared with all the other normal tissues analyzed. While some expression was seen in liver (9.19), all other normal tissues had an expression level of 1 or lower. The absolute numbers in Table 5 were obtained analyzing pools of samples of a particular tissue from different individuals. They cannot be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 6. The absolute numbers depicted in Table 6 are relative levels of expression of Pro124 in 63 pairs of matching and 35 unmatched samples. All the values are compared to normal testis (calibrator). A matching pair is formed by obtaining mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual. 6 TABLE 6 Relative Levels of CSG Pro124 Expression in Individual Samples Matching Normal Sample ID Tissue Cancer Adjacent Pro53P Prostate 1-Normal 472.77 Pro73P Prostate 2-Normal 251.79 Pro77P Prostate 3-Normal 494.56 ProC153 Prostate 4-Normal 0.32 Pro12B Prostate 5-Cancer 674.19 55.88 Pro78XB Prostate 6 109.53 55.54 Pro84XB Prostate 7 1770.5 522.96 Pro109XB Prostate 8 41.38 47.44 Pro101XB Prostate 9 1197.5 362.18 Pro91X Prostate 10 476.13 72.55 Pro13XB Prostate 11 3.28 15.96 Pro125XB Prostate 12 70.08 17.78 Pro23B Prostate 13 201.04 83.3 Pro90XE Prostate 14 424.38 849.41 Pro588P Prostate 15 120.28 Pro34B Prostate 16 553.04 217.14 Pro110 Prostate 17 46.63 326.02 Pro65XB Prostate 18 639.72 2488.9 Pro20XB Prostate 19 61.18 Pro69XB Prostate 20 30.48 4.26 Pro326 Prostate 21 59.53 10.38 ProC215 Prostate 22 525.68 ProC234 Prostate 23 35.13 ProC280 Prostate 24 1265.06 Pro10R Prostate 25 81.71 (Prostatitis) Pro20R Prostate 26 194.19 (Prostatitis) Pro258C Prostate 27 (BPH) 506.95 Pro263C Prostate 28 (BPH) 751.79 Pro267A Prostate 29 (BPH) 53.04 Pro271A Prostate 30 (BPH) 25.46 Pro460Z Prostate 31 (BPH) 102.48 ProC032 Prostate 32 (BPH) 353.51 Pro10P Prostate 33 (BPH) 287.71 Pro13P Prostate 34 (BPH) 19.93 Pro277P Prostate 35 (BPH) 209.54 Pro34P Prostate 36 (BPH) 59.47 Pro705P Prostate 37 (BPH) 13.74 Pro784P Prostate 38 (BPH) 22.05 Pro83P Prostate 39 (BPH) 57.45 Pro855P Prostate 40 (BPH) 392.31 ProC003P Prostate 41 (BPH) 0 ProC034P Prostate 42 (BPH) 6.71 Tst39X Testis 1 1.87 0.19 Kid106XD Kidney 1 0 0 Kid107XD Kidney 2 0 0 Kid109XD Kidney 3 1.1 1.2 Kid10XD Kidney 4 0 0 Kid11XD Kidney 5 0 0 Kid124XD Kidney 6 0.6 0 Kid126XD Kidney 7 10.82 0.03 Kid12XD Kidney 8 0 8.02 Bld32XK Bladder 1 0 0 Bld46XK Bladder 2 1.9 0.9 Bld66X Bladder 3 0 0 BldTR14 Bladder 4 0.27 0 BldTR17 Bladder 5 0.69 0 Lng47XQ Lung 1 2.1 0 Lng60XL Lung 2 0.5 0 Lng75XC Lung 3 0 0 Lng90X Lung 4 0 0 LngAC11 Lung 5 0.81 0.42 Liv15XA Liver 1 2.5 10.9 Liv174L Liver 2 60 230.7 Liv175L Liver 3 144.7 52.3 Liv187L Liver 4 69.88 0.78 Liv201L Liver 5 26.31 53.9 Liv390L Liver 6 6.2 46.02 Liv42X Liver 7 2.73 32.36 Pan71XL Pancreas 1 0.12 0.1 Pan77XL Pancreas 2 138.99 162.06 Pan82XP Pancreas 3 0.41 0 ClnAC19 Colon 1 0.5 0.4 ClnAS12 Colon 2 0 0 ClnAS43 Colon 3 0 0 ClnB34 Colon 4 0.08 0 ClnB56 Colon 5 0 0 ClnDC19 Colon 6 0 0 ClnRC01 Colon 7 0 0 Ovr1005 Ovary 1 1.01 Ovr1028 Ovary 2 1.12 Ovr1040 Ovary 3 0.1 Ovr105O Ovary 4 5.12 Ovr18GA Ovary 5 0 Ovr20GA Ovary 6 0 Ovr230A Ovary 7 0 Ovr233A Ovary 8 0 Mam162X Mammary Gland 1 0 0 Mam14DN Mammary Gland 2 0 0 Mam19DN Mammary Gland 3 0.36 0 End10479 Endometrium 1 4.4 0 End12XA Endometrium 2 2.5 0 End28XA Endometrium 3 0.4 0 End3AX Endometrium 4 0 0 End5XA Endometrium 5 0 0.5 End65RA Endometrium 6 0 0 CvxKS52 Cervix 1 0 0 CvxKS83 Cervix 2 0.12 0.94 CvxNK23 Cervix 3 0 0 0 &equals; Negative In the analysis of matching samples, the higher levels of expression were in prostate, showing a high degree of tissue specificity for prostate tissue. Of all the samples different than prostate analyzed, only liver tissue samples (liver 2 through liver 7) and pancreas tissue sample (pancreas 2) showed an expression comparable to the mRNA expression in prostate. All remaining tissue samples had relative Pro124 mRNA expression levels of less than 10 (except kidney cancer sample 7 that was 10.82). These results confirmed a high degree of tissue specificity as obtained with the panel of normal pooled samples (Table 5). Furthermore, the level of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual were compared. This comparison provides an indication of specificity for the cancer (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Table 6 shows overexpression of Pro124 in 10 out of 15 primary prostate cancer tissues compared with their respective normal adjacent. Thus, there was overexpression in the cancer tissue for 66.66% of the prostate matching samples tested. The mRNA overexpression in 66.66% of the prostate matching cancer samples tested is indicative of Pro124 being a diagnostic marker for prostate cancer.