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Androgen Receptor Mutations in Androgen-Independent Prostate Cancer: Cancer and Leukemia Group B Study 9663

Mary-Ellen Taplin, Barur Rajeshkumar, Susan Halabi, Cary P. Werner, Bruce A. Woda, Joel Picus, Walter Stadler, Daniel F. Hayes, Philip W. Kantoff, Nicholas J. Vogelzang, Eric J. Small

From the University of Massachusetts Memorial Health Center, Worcester, and Dana-Farber Cancer Institute, Boston, MA; Cancer and Leukemia Group B Statistical Center, Durham NC; Washington University Barnes Hospital, St Louis, MO; University of Michigan Comprehensive Cancer Center, Ann Arbor, MI; University of Chicago, Chicago, IL; and University of California at San Francisco, San Francisco, CA.

Address reprint requests to Mary-Ellen Taplin, MD, University of Massachusetts Memorial Health Center, Department of Oncology, 55 Lake Ave North, Worcester, MA 01655; email: taplinm{at}ummhc.org.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
Purpose: The mechanisms responsible for prostate cancer androgen independence are diverse. Mutations of the androgen receptor (AR) gene that broaden ligand specificity have been implicated. Bone marrow specimens containing prostate tumor were obtained from men undergoing antiandrogen withdrawal for AR sequence analysis and clinical correlation.

Materials and Methods: Eligible men enrolled on a trial of antiandrogen withdrawal had a minimum prostate-specific antigen (PSA) level of 5 ng/dL that was increasing on castration therapy including an antiandrogen. With informed consent, marrow biopsies were obtained to collect prostate tumor. Additional samples were obtained from men enrolled on chemotherapy trials. AR cDNA or DNA was polymerase chain reaction–amplified, cloned, and sequenced. The AR CAG repeat length was recorded.

Results: One hundred eighty-four bone marrow biopsies were obtained, and 48 had prostate tumor detected by light microscopy. The ARs from these 48 samples were sequenced. Overall, five (10%) of 48 tumors had mutated ARs. AR point mutations were detected in the hormone-binding domain involved in transcription factor binding. Three mutations were novel in prostate cancer. One tumor sample had a CAG repeat length of 21, compared with germline length of 22 repeats. There was no association between detectability of AR mutations and antiandrogen withdrawal response or survival.

Conclusion: These data suggest that AR mutations are present in approximately 10% of patients with prostate cancer who experience treatment failure with hormone therapy that included an antiandrogen. Mutations in the AR likely confer a growth advantage for a subset of progressive prostate cancers. Correlation of AR mutation with antiandrogen withdrawal response or survival could not be made.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
DESPITE RECENT investigations, the mechanisms responsible for the development of androgen-independent prostate cancer (AiPC) are unclear. Advanced prostate cancer is most often treated with hormone therapy. Androgen deprivation initiates apoptosis in the majority of prostate cancer cells.¹ Approximately 80% of patients will respond favorably to castration therapy; however, despite a wide range of response duration, the average response is approximately 18 months.² Antiandrogens are commonly prescribed either as part of initial androgen deprivation or as sequential hormone therapy.³ A clinical response to discontinuation of antiandrogen has been noted in approximately 30% of patients treated with hydroxyflutamide.⁴ Additionally, there are case reports of withdrawal response to other therapies, including bicalutamide, nilutamide, diethylstilbestrol, megestrol acetate, cyperone acetate, and estramustine.⁵ It has been proposed that the antiandrogen withdrawal response results from mutated androgen receptors (ARs).

The AR, a ligand-activated nuclear transcription factor, is central to the growth of prostate cancer and its response to hormone therapy. AR protein and AR-regulated genes such as prostate-specific antigen (PSA) remain expressed in AiPC, suggesting that AR continues to play an important signaling role.^6,7 AR alterations, including downregulation, amplification, and triplet repeat or point mutations, have been described in prostate cancer.^8–10 The significance of these AR changes in the development of AiPC remains under investigation.

The true incidence of AR point mutations in clinical prostate cancer has been difficult to assess. They seem to be rare in early-stage untreated prostate cancer^11,12 and more common in advanced AiPC.^10,13 Factors that make it difficult to determine the in vivo frequency of AR mutations include patient selection, tumor heterogeneity, tissue source (prostate gland v metastases), method of tissue preservation, and molecular methods.¹² The AR gene mutation database and a recent review list reported AR mutations from prostate cancer and androgen insensitivity syndrome.^14,15

In general, AR mutations in AiPC occur in regions that affect AR function by altering ligand interaction or increasing transcriptional activity. Transfection experiments revealed that AR point mutations in the hormone-binding domain (HBD) allow activation by ligands other than testosterone, including antiandrogens.¹⁶ The AR from the prostate cancer cell line LNCaP (Thr877Ala mutation) was activated by progesterone, estradiol, and the antiandrogens cyproterone acetate, anandron, and hydroxyflutamide.¹⁷ AR mutations from clinical tumor samples were activated by progesterone (Val715Met, Thr877Ser, His874Tyr), estradiol (Thr877Ser, His874Tyr), adrenal androgens (Val715Met), hydrocortisone (Leu701H/T877Ala), and hydroxyflutamide (Val715Met, Thr877Ser, His874Tyr, Asp890Asn).^16,18,19 These data imply that androgen signaling remains intact and that selection of cells harboring altered AR with broadened ligand specificity occurs in AiPC.

We report results of Cancer and Leukemia Group B (CALGB) Study 9663, which was undertaken to determine the following: (1) the feasibility of collecting bone marrow biopsies from prostate cancer patients in a cooperative group setting, (2) the incidence of marrow invasion by prostate tumor in random bone marrow biopsy, (3) the influence of previous prostate radiation on obtaining prostate tumor, (4) the frequency and type of AR mutations, and (5) association of AR mutations with response to antiandrogen withdrawal. The results reported reflect the largest multi-institutional prospective collection of metastatic prostate cancer with sequencing the entire AR gene.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
Patients
The CALGB conducted a phase III study (CALGB 9583) in which 260 men with AiPC were randomly assigned to antiandrogen withdrawal together with simultaneous ketoconazole and hydrocortisone versus antiandrogen withdrawal alone, followed by sequential ketoconazole and hydrocortisone. Metastatic disease with progression despite castrate levels of testosterone, prior antiandrogen therapy for a minimum of 4 weeks, and a minimum PSA level of 5 ng/mL were required; treatment with sequential antiandrogens was allowed. No prior chemotherapy was allowed. Bone marrow biopsies were obtained from 164 patients enrolled on CALGB 9583 and from 20 patients enrolled on CALGB chemotherapy trials (CALGB 9480, 9680, 9780).

Tissue
CALGB 9663 (AR mutations in hormone-refractory prostate cancer) was a mandatory companion trial to CALGB 9583. Written informed consent was obtained to perform a bone marrow aspirate and biopsy from the posterior iliac crest before antiandrogen withdrawal. Investigators were provided with an instructional video for sample procurement and shipment to a central laboratory. Marrow core biopsies were immediately cut in half; the inner half was snap frozen, placed in OCT (Tissue-tek; VWR International, West Chester, PA), and shipped overnight on dry ice. Samples were stored at -80°C until analysis. The outer half was placed in formalin and shipped for routine pathologic evaluation. A hematopathologist (B.A.W.) quantified marrow prostate cancer, and positive samples (n = 48) were used for AR analysis. Peripheral blood and marrow aspirate were collected and cryopreserved in liquid nitrogen at a central laboratory.

Reverse Transcriptase Polymerase Chain Reaction Analysis of AR Transcripts
Previous work demonstrated that AR amplified from marrow biopsies containing prostate tumor is from the prostate tumor and not from contaminating hematopoietic cells.¹³ Biopsy RNA was extracted from 10 consecutive 10-µm frozen sections using Rnazol-B (TM Cinna Scientific, Friendswood, TX), and cDNA was synthesized with MMTV reverse transcriptase and random hexamer primer. cDNA integrity was evaluated by polymerase chain reaction (PCR) amplification of beta-2 microglobulin.²⁰ The AR was sequenced in five portions: A exon codons 1–53, 93–443, 431–520 and exons B-H codon 493–3'UTR (Fig 1). The CAG repeat length (starting at codon 58) was measured separately (see below). Primer sequences used for PCR are shown in Table 1. PCR reactions were done in 50-µL aliquots containing 1 µL of Taq DNA polymerase (Perkin Elmer; Applied Biosystems, Foster City, CA), 10 x PCR buffer, 2.5 mmol/L of dNTP, 100 ng of each primer, 100 mmol/L of Mg, and 50 to 100 ng of cDNA or DNA. PCR conditions included 94°C denaturation for 20 seconds, 55° to 65°C annealing for 30 seconds, optimized for primer sequence, 72°C extension for 90 seconds, and cycle length between 25 and 40 seconds. Dimethyl sulfoxide was included in exon A PCR reactions. Negative controls in which the reverse transcriptase step was omitted were routinely included. PCR products were cloned according to previously reported methods,¹³ and five clones were sequenced using an ABI Prism 373 fluorescent DNA sequencer (Perkin Elmer). Base changes were determined to be mutations rather than Taq polymerase errors on the basis of identification in multiple plasmids and isolation from a second independent PCR amplification and analysis. AR was sequenced from peripheral-blood lymphocyte DNA to exclude genomic polymorphism. Biopsies that contained more than 90% tumor cells were sequenced using genomic DNA rather than cDNA.

View larger version (20K): [in this window] [in a new window]   Fig 1. Structure of the androgen receptor and primers used for polymerase chain reaction amplification. Exons A through H and the locations of the transactivation, DNA-binding, and hormone-binding domains are shown. The positions and orientations of the primers used are also shown. The AR was sequenced in five portions.

CAG Repeat Measurement
AR primers (Table 1) spanning the CAG repeat were end labeled with gamma phosphorus-32 adenosine triphosphate for PCR. PCR reactions included primers, 1 µL of cDNA from tumor or 1 µL of DNA from matching peripheral-blood lymphocytes, Taq polymerase, and 38 cycles with denaturing 94°C for 60 seconds, annealing 60°C for 60 seconds, and extension 72°C for 60 seconds. PCR products together with a ladder (CAG repeats from 14–26) were run on sequencing gels (1,800 V, 3 hours), and repeat length was calculated from auto radiographs.

Statistical Analysis
The proportion of patients with AR expression and 95% confidence intervals (CIs) based on the binomial distribution were computed. Fisher’s exact test was used to correlate the AR mutation status with objective response (defined as complete response or partial response) and 50% (or 75%) decline in PSA (PSA decline was defined as two consecutive declines in PSA, 4 weeks apart). The Kaplan-Meier product-limit method was used to estimate the overall survival and progression-free survival by the AR mutation status. Survival duration was defined as the time between randomization (or study entry) and death. Patients were censored if they were known to be alive or if they were lost-to follow-up. Progression-free survival was defined as the time between randomization (or study entry) and disease progression or death, whichever occurred first. For patients with measurable disease, progression was defined as the date of the first computed tomography scan that showed new lesions with a greater than 25% increase in the sum of the perpendicular diameters of previously measured disease. For patients with elevated serum PSA levels (with either nonmeasurable but assessable or measurable disease), progressive disease was defined as two consecutive increases in PSA, at least 2 weeks apart, each greater than 50% above the nadir or the pretreatment baseline, whichever is lowest.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
Patient Characteristics
The characteristics of patients with a positive biopsy for tumor are listed in Table 2. The median age was 72 years (interquartile range, 65 to 78 years). Ethnic distribution included 74% white, 24% African-American, and 2% other. Fifty-three percent with a positive biopsy had Gleason 8 to 10 tumor at diagnosis. Ninety-eight percent had documented bone metastases. The median PSA was 156 ng/mL (interquartile range, 42 to 509 ng/mL). Antiandrogen therapy before enrollment was 48% flutamide, 40% bicalutamide, 6% nilutamide, and 2% both bicalutamide and flutamide. There were 137 patients with no tumor detected in marrow biopsy. Compared with the patients with a positive biopsy, these patients had higher performance status (3% with performance status of 2 v 17% with performance status of 2), fewer positive bone scans (78% v 98%), fewer Gleason 8 to 10 scores (43% v 52%), higher median hemoglobin level (13 g/dL v 11 g/dL), lower median PSA (44 ng/mL v 167 ng/mL), lower median alkaline phosphatase (116 U/L v 258U/L), and lower lactate dehydrogenase (207 U/L v 254 U/L).

View larger version (95K): [in this window] [in a new window]   Fig 2. (A,C) Direct sequencing of reverse transcriptase polymerase chain reaction products from patient no. 66850 and patient no. 71766 demonstrating codon 877 and 879 mutations. Wild-type sequence is below the panel and the amino acid encoded by mutant AR is above. (B,D) Representative sections from marrow biopsy demonstrating tumor used for RNA extraction.

CAG Repeat Length Analysis
Subject CAGs ranged from 16 to 28 repeats. The average CAG length for the group was 20.93. The average for African-American patients was 21.25 (n = 8), and the average for white patients was 20.78 (n = 19). There was one somatic alteration in CAG length with 21 repeats (tumor) and 22 repeats (genomic) (95% CI, 0.0 to 19.0).

Clinical Correlation
Patient responses to antiandrogen withdrawal, survival, and progression free survival were analyzed according to AR mutation status. There was a 20% PSA response to antiandrogen withdrawal (> 50% decline) in the AR mutations group, compared with a 7% PSA response in the no mutation group (95% CI, 2% to 21%). The median survival in the AR mutation group was 9.2 months, compared with 11.2 months in the no mutation group (95% CI, 8.0 to 14 months). The progression-free survival was 3.3 months in the AR mutation group, compared with 3.1 months in the no mutation group (95% CI, 2.1 to 4.1 months).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
We have shown that it is feasible to collect prostate cancer from the bone marrow of men with AiPC. Twenty-five percent of patients had tumor that could be investigated for AR changes, and 10% of these had AR mutations. These data are unique because fresh metastatic prostate tumor was collected prospectively in the context of a multi-institutional trial, thereby reducing selection biases. The use of fresh tissue reduces problems with cDNA/DNA quality inherent in the use of archival tissue.¹² Metastatic prostate tumor samples are notoriously difficult to obtain for correlative study. It has previously been demonstrated that bone marrow is a good source of metastatic prostate tumor, but the yield of obtaining tumor from a single random biopsy was not known.¹³ The frequency of obtaining tumor visible by light microscopy will depend on the extent and location of metastases and the placement and size of the bone marrow biopsy. Because we wanted to study samples enriched with tumor, we required prostate cancer microscopically present rather than detected by PCR alone.

Patients from CALGB 9583 had a minimum PSA level of 5 ng/dL and a median PSA level of 58 ng/dL, and 164 marrow biopsies were collected to obtain 48 with prostate tumor. These patients had not had multiple prior second-line hormone therapies or chemotherapy. Positive biopsies were more likely to be obtained from men who had more advanced disease, as reflected by lower performance status, lower hemoglobin, and higher PSA, alkaline phosphatase, and lactate dehydrogenase. Patients with a positive biopsy had a median PSA level of 156 ng/mL, compared with a median PSA level of 58 ng/mL for all enrolled patients (Table 2 and data not shown). Prior radiation to the prostate or pelvis was also a factor in obtaining useful samples. The yield of marrow tumor was twice as likely if patients had not received prior radiotherapy. Although patients and participating physicians accepted the biopsy procedure and there were no adverse events, it is labor intensive to obtain meaningful sample sizes in patients with low-volume disease. Bone marrow is a good source of prostate tumor for correlative study. The design of future studies hinging on marrow biopsies should target patients described in Table 2. In addition, the tumor yield could be increased by directing biopsies to suspicious areas on bone scan, which was not a requirement of this study.

Our results demonstrate that somatic AR mutations were found in five (10%) of 48 chemotherapy-naive AiPC patients. Tumor samples before initiation of hormone therapy were not available for comparison. Previous analyses of bone marrow samples by the author (M.-E.T.) have described AR mutations in 30% to 50% of analyzed prostate tumors.^10,13 The lower frequency of AR mutations from this subject population likely reflects an earlier-disease stage and shorter duration of antiandrogen therapy, which became the clinical standard during the course of the trial.

Other reports of AR mutations in prostate cancer used tissue from the gland or regional lymph nodes in systemically untreated patients.^11,12,22 With the exception of one report,²² AR mutations were uncommon in early prostate cancer. The largest series found no mutations in 99 prostate samples and 11 mutations (exons B-H) in eight of 38 regional lymph nodes (8% of all samples analyzed and 21% of regional lymph nodes).¹² Tilley et al²² reported mutations in 11 (44%) of 25 prostate tumors from the gland before systemic therapy. A possible explanation for the higher incidence of mutations observed by Tilley et al is selection of biologically aggressive tumors. In the Tilley cohort, 40% progressed rapidly to AiPC; there were 75% mutations in these patients, compared with 25% in patients who responded for BORDER="0"> 2 years of hormone therapy.²²

Despite case reports or small series describing AR mutations in AiPC, the frequency and functional significance of mutations has been difficult to assess.¹⁵ The only other prospectively collected samples (n = 13) were from a trial of bicalutamide (150 mg) as a second-line hormone therapy.²³ AR mutations (codons 877, 890) were found in five (38%) of 13 patients.¹⁰ Patients whose tumor harbored an AR mutation were more likely to have been previously treated with several years of hydroxyflutamide and were more likely to respond to second-line bicalutamide therapy.¹⁰ The majority of published reports, including the present data set, are based on a single biopsy, so an assessment of tumor heterogeneity regarding AR mutations is not available. Our results together with the literature suggest that point mutations in the AR occur in 10% to 40% of prostate tumors depending on the stage, type, and duration of previous hormone (antiandrogen) therapy and the composition of AiPC cells.

The transactivation domain (exon A) encodes 58% of the AR coding region and the importance of including this region in analysis has been emphasized.²² The majority of AR sequence analyses in prostate cancer since 1992 did not include the transactivation domain. A major contribution of our data is that this is the first large evaluation of the AR transactivation domain from metastatic prostate samples. Although our initial analysis (data not shown) demonstrated five mutations between codons 198 and 390, we were not able to confirm these mutations. It is possible that these mutations exist in a minor population of cells or that when the biopsy was recut to confirm the mutations, tumor was lost. Nevertheless, we cannot conclude that mutation of the transactivation domain is a high frequency event as suggested by Tilley et al.²²

The HBD is highly conserved among steroid hormone receptors, and previous AR mutations in this region broadened ligand specificity, including activation by antiandrogens.^16–19 Three of our mutations (877, 879) were in the AF-2 domain of exon H. The AF-2 domain spans codons 872 to 910, binds coregulatory proteins, including transcriptional intermediary factor 2, and interacts with the transactivation domain.²¹ This region has been a hot spot for AR mutations in prostate cancer. The recent description of the crystal structure of the wild-type and Thr877Ala mutant AR HBD demonstrated how the Thr877Ala mutation changes the binding pocket to allow binding of progesterone and other nonclassic ligands.²⁴ Thus HBD mutations that are selected in the context of antiandrogen treatment confer a cellular growth advantage and allow activation by ligands other than androgens.

We report two additional unique mutations in prostate cancer located in exon E (codons 741, 756). These mutations fall outside the three areas where prostate cancer AR mutations have clustered, and future functional analyses of these mutations will increase our understanding of ligand and coactivator/repressor binding.²⁵ We did not detect any AR transcripts with more than one mutation. Prostate cancers containing multiple AR mutations^2–4 have been previously reported in nine tumors.^{10,13,19,22,26,27} The functional consequences of double amino acid changes will depend on how these substitutions affect protein interactions within the AR and with accessory proteins and remain under study.

The mutant AR clones were present in 80% to 100% of the clones from each tumor sample, suggesting that these mutations provide a significant growth advantage to tumor cells. Because all patients had been previously treated with antiandrogens, these data do not address the issue of what role antiandrogens themselves have in selecting mutant AR. Previous work has suggested that mutated AR is uncommon in patients not exposed to antiandrogen therapy and that AR mutations from patients treated with hydroxyflutamide were not activated by bicalutamide.^10,16 This is the first study to systemically evaluate AR mutations from patients treated with bicalutamide. However, in this series in which many patients received bicalutamide, it seems that AR mutations occur with similar frequency regardless of the type of antiandrogen exposure.

Association of the AR sequence data with clinical parameters reveals that there was no relationship of AR mutations with response to antiandrogen withdrawal. A possible association may not have been detected, however, given the small sample size. Alternatively, AR mutations may not be the cause of the antiandrogen withdrawal syndrome. An alternative explanation for the antiandrogen withdrawal response could be an aberrant interaction between an AR coregulatory protein and antiandrogen. This hypothesis was not tested but will be the subject of future investigation. Additionally, there was no association between AR mutation and survival, type of antiandrogen used, or initial duration of response to hormone therapy.

One (3%) of 27 prostate cancer samples had a somatic contraction of the CAG repeat length, 21 repeats in tumor and 22 repeats in genomic DNA. Somatic alterations of the CAG repeat length seem to be a rare event in prostate cancer. Two additional microsatellite contractions have been reported from 97 samples analyzed by three groups, CAG₂₀ CAG_18, and CAG₂₄CAG₁₈.^9,22,28 The CAG repeats in our patients ranged from 16 to 28, with a mean of 20.93. Twelve samples from white patients (n = 19) had CAG repeats less than 22, which is the average repeat length for white men.²⁹ It remains under investigation whether short CAG repeat is a risk factor for prostate cancer or aggressive phenotype.

In conclusion, we describe a comprehensive analysis of AR sequence, including the transactivation domain and CAG repeat length from bone marrow metastases from AiPC patients. AR point mutations were found in 10% of samples, and CAG repeat mutations were found in 3%. AR mutations were found in the HBD and were concentrated in the AF-2 region central to transcription factor binding. In accordance with previous analyses, AR mutations likely result from selective pressure secondary to antiandrogens, including bicalutamide, and result in broadened ligand specificity.^10,13,16 No correlation of AR mutations with antiandrogen withdrawal or survival could be made because of small sample size related to difficulty in collecting tumor from patients with low-volume disease.

	APPENDIX

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX REFERENCES

 
The following institutions and individuals participated in the study and many received support from the National Cancer Institute: CALGB Statistical Office, Durham, NC (Stephen George, PhD, supported by grant no. CA33601); Dartmouth Medical School-Norris Cotton Cancer Center, Lebanon, NH (Marc Ernstoff, MD, supported by grant no. CA04326); Georgetown University Medical Center, Washington, DC (Edward P. Gelmann, MD, supported by grant no. CA77597); Mount Sinai School of Medicine, New York, NY (Lewis Silverman, MD, supported by grant no. CA04457); Rhode Island Hospital, Providence, RI (William Sikov MD, supported by grant no. CA08025); State University of New York Upstate Medical University, Syracuse, NY (Stephen L. Graziano, MD, supported by grant no. CA21060); Ohio State University, Columbus, OH (Clara Bloomfield, MD, supported by grant no. CA77658); University of California at San Diego, San Diego, CA (Stephen Seagren, MD, supported by grant no. CA11789); University of California at San Francisco, San Francisco, CA (Alan Venook, MD, supported by grant CA60138); University of Chicago Medical Center, Chicago, IL (Gini Fleming, MD, supported by grant no. CA41287); University of Illinois at Chicago, Chicago, IL (David Gustin, MD; supported by grant no. CA74811); University of Maryland Cancer Center, Baltimore, MD (David Van Echo, MD, supported by grant no. CA31983); University of Massachusetts Medical Center, Worcester, MA (Mary-Ellen Taplin, MD, supported by grant no. CA37135); University of Minnesota, Minneapolis, MN (Bruce A. Peterson, MD, supported by grant no. CA16450); University of Missouri/Ellis Fischel Cancer Center, Columbia, MO (Michael C. Perry, supported by grant no. CA12046); University of Nebraska Medical Center, Omaha, NE (Anne Kessinger, MD, supported by grant no. CA77298); University of Tennessee Memphis, Memphis, TN (Harvey B. Niell, MD, supported by grant no. CA47555); Vermont Cancer Center, Burlington, VT (Hyman B. Muss, MD, supported by grant no. CA77406); Wake Forest University School of Medicine, Winston-Salem, NC (David D. Hurd, MD, supported by grant no. CA03927); and Washington University School of Medicine, St Louis, MO (Nancy Bartlett, MD, supported by grant no. CA77440).

	ACKNOWLEDGMENTS

 
We thank Dr Edward Gelmann for providing support in the development of this protocol, Dr Shuk Mei Ho and Dr Kin-Mang Lau for providing the genomic ladder to measure CAG repeat length, and Dr Steven Balk for his guidance.

	NOTES

 
M.-E.T supported by grant nos. U10 CA78967 and U10CA31946 from the National Cancer Institute, Bethesda, MD.

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Submitted November 19, 2002; accepted April 29, 2003.

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				P. Mathew, P. F. Thall, C. D. Bucana, W. K. Oh, M. J. Morris, D. M. Jones, M. M. Johnson, S. Wen, L. C. Pagliaro, N. M. Tannir, et al. Platelet-Derived Growth Factor Receptor Inhibition and Chemotherapy for Castration-Resistant Prostate Cancer with Bone Metastases Clin. Cancer Res., October 1, 2007; 13(19): 5816 - 5824. [Abstract] [Full Text] [PDF]

				M. C. Hodgson, I. Astapova, A. N. Hollenberg, and S. P. Balk Activity of Androgen Receptor Antagonist Bicalutamide in Prostate Cancer Cells Is Independent of NCoR and SMRT Corepressors Cancer Res., September 1, 2007; 67(17): 8388 - 8395. [Abstract] [Full Text] [PDF]

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				Y. B. Wetherill, J. K. Hess-Wilson, C. E.S. Comstock, S. A. Shah, C. R. Buncher, L. Sallans, P. A. Limbach, S. Schwemberger, G. F. Babcock, and K. E. Knudsen Bisphenol A facilitates bypass of androgen ablation therapy in prostate cancer Mol. Cancer Ther., December 1, 2006; 5(12): 3181 - 3190. [Abstract] [Full Text] [PDF]

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