Expression studies and mutational analysis of the androgen regulated homeobox gene NKX3.1 in benign and malignant prostate epithelium.

PURPOSE: The NKX-3.1gene is an androgen regulated prostate specific homeobox gene that is believed to have a vital role in normal prostate development. In mice the homologue NKx3.1 is exclusively expressed in prostate epithelium. In humans NKX3.1 expression is also restricted to the prostate but to our knowledge the cellular location has not been described. Furthermore, since NKX3.1 maps to chromosomal band 8p21, a region with high loss of heterozygosity in prostate cancer, the gene has been proposed to have tumor suppressor function. In this study we demonstrate that in human prostates NKX3.1 is expressed exclusively in secretory epithelial cells and the level of NKX3.1 expression remains invariant in normal tissue and in tissue showing various grades of prostate cancer. In the 19 cases examined the DNA sequences of the NKX3.1 gene were identical and no mutation was detected. MATERIALS AND METHODS: Frozen tissue from patients who underwent radical prostatectomy was used for this study. For in situ hybridization experiments a 377 bp fragment corresponding to a portion of the 3' untranslated region of the NKX3.1 gene was amplified by polymerase chain reaction and cloned into the pCRII plasmid vector Invitrogen. Antisense or sense [33P] uridine triphosphate labeled RNA probes were generated with SP6 or T7 RNA polymerase and hybridized to the tissue sections. Slides were exposed to photographic emulsion and visualized on autoradiography. Laser capture microdissection was performed to procure pure populations of malignant epithelium. DNA was isolated by digesting samples in proteinase K buffer. Polymerase chain reaction and direct sequencing was performed using standard protocols. RESULTS: In vitro hybridization showed that NKX3.1 expression was restricted to secretory epithelial cells within benign prostate glands. No expression was detected in stroma or infiltrating lymphocytes. NKX3.1 was expressed in all grades of malignant epithelium in all 25 cases examined. Direct sequencing of the coding region of NKX3.1 revealed the wild-type sequence in all 18 microdissected cancers analyzed. CONCLUSIONS: Based on our studies we propose that NKX3.1 gene expression is restricted to benign and malignant secretory epithelium within the prostate but NKX3.1 does not appear to be a classic tumor suppressor gene responsible for prostate cancer initiation. These findings are consistent with the role of NKX3.1 in the development of normal prostate epithelium and maintenance of normal secretory function. Thus, NKX3.1 may represent a useful molecular marker for benign and malignant prostate epithelium.

How to improve prostate biopsy detection of prostate cancer.

The combination of serum prostate-specific antigen (PSA) testing and transrectal ultrasonography is a highly effective strategy to diagnose prostate cancer at an early curable stage. Even though PSA is the most useful serum biomarker to aid in prostate cancer detection, it has limited specificity: as many as 75% of men who undergo prostate biopsy because of an elevated PSA do not have prostate cancer. Additionally, sextant prostate biopsies miss prostate cancer at least 20% of the time. To reduce the number of false-negative biopsies, many have advocated obtaining 12 or more cores in a single biopsy session. Studies have shown that this practice is safe and can enhance cancer detection modestly. Although it is unlikely that prostate cancer imaging will replace prostate biopsy in the near future, many exciting new imaging technologies should eventually improve targeting of prostate needle biopsy and reduce false-negative biopsies. Some of the most exciting areas include power Doppler sonography, microbubble intravenous ultrasound contrast agents, and magnetic resonance spectroscopy. These functional imaging modalities can assess tumor blood flow and metabolic activity at a cellular level and can detect malignant changes that may not be detected by standard anatomic imaging.

Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma.

Annexin I protein expression was evaluated in patient-matched longitudinal study sets of laser capture microdissected normal, premalignant, and invasive epithelium from human esophageal squamous cell cancer and prostatic adenocarcinoma. In 25 esophageal cases (20 by Western blot and 5 by immunohistochemistry) and 17 prostate cases (3 by Western blot and 14 by immunohistochemistry), both tumor types showed either complete loss or a dramatic reduction in the level of annexin I protein expression compared with patient-matched normal epithelium (P < or = 0.05). Moreover, by using Western blot analysis of laser capture microdissected, patient-matched longitudinal study sets of both tumor types, the loss of protein expression occurred in premalignant lesions. Concordance of this result with immunohistochemical analysis suggests that annexin I may be an essential component for maintenance of the normal epithelial phenotype. Additional studies investigating the mechanism(s) and functional consequences of annexin I protein loss in tumor cells are warranted.

A prospective randomized trial comparing 6 versus 12 prostate biopsy cores: impact on cancer detection.

PURPOSE: Several studies suggest that sextant transrectal ultrasound guided biopsy of the prostate provides insufficient material to detect all clinically important prostate cancer, and obtaining more biopsy cores may improve the cancer detection rate. We performed a prospective randomized trial comparing 6 to 12 prostate biopsy cores to determine the impact on the cancer detection rate. MATERIALS AND METHODS: We prospectively randomized 244 men, including 71 (29%) black men, with a mean age plus or minus standard deviation of 65 +/- 8 years to undergo biopsy with 6 or 12 peripheral zone tissue cores. In our study subjects serum total prostate specific antigen (PSA) was between 2.5 and 20 ng./ml., and/or digital rectal examination was suspicious for cancer. All men completed a self-administered pre-biopsy and 2 post-biopsy questionnaires at 2 and 4 weeks. Cancer detection rates were compared in the groups and correlated with race, biopsy history, digital rectal examination findings, total PSA, transrectal ultrasound volume and PSA density, as determined by the formula, total PSA/transrectal ultrasound volume. RESULTS: The cancer detection rate in the 6 and 12 core groups was almost identical (26% and 27%, p = 0.9). There was no significant difference in cancer detection in the 2 trial arms with respect to subject race, biopsy history, digital rectal examination findings, total PSA, transrectal ultrasound volume or PSA density. However, our study did not have the statistical power to rule out small differences. CONCLUSIONS: The overall cancer detection rate is not materially increased by 12 core, peripheral zone biopsy in men in whom prostate cancer was mainly detected by screening.

Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines.

Specific populations of normal and malignant epithelium from three radical prostatectomy tissue specimens were procured by laser capture microdissection (LCM) and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Six proteins that were only seen in malignant cells and two proteins that were only seen in benign epithelium were reproducibly observed in two of two cases examined. Furthermore, these proteins were not observed in the 2-D PAGE profiles from the patient-matched microdissected stromal cell populations, but were seen in the protein profiles from the undissected whole cryostat sections. One of these proteins was determined to be prostate-specific antigen (PSA) by Western blot analysis, and intriguingly the remaining protein candidates were found to be at least as abundant as the PSA protein. Comparison of 2-D PAGE profiles of microdissected cell with matched in vitro cell lines from the same patient, and metastatic prostate cancer cell lines (LnCaP and PC3) showed striking differences between prostate cells in vivo and in vitro with less than 20% shared proteins. The data demonstrate that 2-D PAGE analysis of LCM-derived cells can reliably detect alterations in protein expression associated with prostate cancer, and that these differentially expressed proteins are produced in high enough levels which could allow for their clinical utility as new targets for therapeutic intervention, serum markers, and/or imaging markers.

Geographic variation in patient surveillance after radical prostatectomy.

BACKGROUND: Prostate cancer is often diagnosed early enough in its clinical course to permit radical prostatectomy to be done with curative intent, yet many patients experience tumor recurrence. Most patients receive postoperative surveillance, but the intensity of testing varies appreciably. We sought to evaluate the influence of geographic location on the variability of surveillance intensity. METHODS: Questionnaires pertaining to postoperative surveillance were mailed to 4467 members of the American Urological Association (AUA). Practice pattern variation was assessed among 24 large metropolitan statistical areas, among nine United States census regions, and by health maintenance organization penetration rate. RESULTS: Of 4467 urologists surveyed, 1416 (32%) responded and 1050 (24%) responses were evaluable. Correlation analysis showed that mean follow-up intensity across modalities surveyed was highly correlated across tumor, node, metastasis (TNM) stages and years postsurgery. We found no significant main effects attributable to metropolitan statistical area, United States (US) census region, or health maintenance organization (HMO) penetration rate for commonly used surveillance modalities: serum prostate-specific antigen (PSA), office visit, and urinalysis. For infrequently used modalities, there were minimal effects on testing intensity of US census region, metropolitan statistical area, and HMO penetration rate. Few two-way and three-way interactions were significant. CONCLUSIONS: The utilization of commonly used surveillance modalities by urologists caring for patients after radical prostatectomy is not affected by metropolitan statistical area, US census region, or HMO penetration rate.

How tumor stage affects american urologists' surveillance strategies after prostate cancer surgery.

The factors which influence decision-making among urologists are not well understood. We evaluated how tumor stage in patients subjected to potentially curative surgery for carcinoma of the prostate affects the self-reported follow-up strategies employed by practicing United States urologists. Standardized patient profiles and a detailed questionnaire based on these profiles were mailed to 4,467 randomly selected members of the American Urological Association (AUA), comprising 3,205 US and 1,262 non-US urologists. The effect of TNM stage on the surveillance strategies chosen by respondents was analyzed by repeated-measures ANOVA. There were 1, 050 respondents who provided evaluable data of whom 760 were from the US. The three most commonly used surveillance modalities by urologists were office visit, serum PSA level, and urinalysis. Nine of the 11 most commonly requested modalities were ordered significantly (p<0.001) more frequently with increasing TNM stage. This effect persisted through 10 years of follow-up, but the differences across stage were tiny. Fifty-five percent of US respondents do not modify their strategies at all according to the patient's TNM stage. Most American AUA members performing surveillance after potentially curative radical prostatectomy for otherwise healthy patients use the same follow-up strategies irrespective of TNM stage. These data permit the rational design of a randomized clinical trial of two alternate follow-up plans. The two trial arms would employ office visits, blood tests, and urinalyses at different frequencies based on current actual practice patterns; there would be no imaging tests in either arm.

An approach to proteomic analysis of human tumors.

A strategy for proteomic analysis of microdissected cells derived from human tumor specimens is described and demonstrated by using esophageal cancer as an example. Normal squamous epithelium and corresponding tumor cells from two patients were procured by laser-capture microdissection and studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Fifty thousand cells resolved approximately 675 distinct proteins (or isoforms) with molecular weights ranging between 10 and 200 kDa and isoelectric points of pH 3-10. Comparison of the microdissected protein profiles showed a high degree of similarity between the matched normal-tumor samples (98% identical). However, 17 proteins showed tumor-specific alterations, including 10 that were uniquely present in the tumors and seven that were observed only in the normal epithelium. Two of the altered proteins were characterized by mass spectrometry and immunoblot analysis and were identified as cytokeratin 1 and annexin I. Acquisition of 2D-PAGE protein profiles, visualization of disregulated proteins, and subsequent determination of the identity of selected proteins through high-sensitivity MS-MS microsequencing are possible from microdissected cell populations. These separation and analytical techniques are uniquely capable of detecting tumor-specific alterations. Continued refinement of techniques and methodologies to determine the abundance and status of proteins in vivo holds great promise for future study of normal cells and associated neoplasms. Mol.

Characterization of intracellular prostate-specific antigen from laser capture microdissected benign and malignant prostatic epithelium.

The proportion of unbound serum prostate-specific antigen (PSA; percent-free PSA) is reported to be lower in men with prostate cancer compared to men with benign prostates (U. H. Stenman et al., Cancer Res., 51: 222-226, 1991; H. Lilja et al., Clin. Chem., 37: 1618-1625, 1991; D. L. Woodrum et al., J. Urol., 159: 5-12, 1998; W. J. Catalona et al., J. Am. Med. Assoc., 279: 1542-1547, 1998). The majority of immunoreactive PSA in serum is complexed to alpha-1-antichymotrypsin (ACT). Two major mechanistic questions have previously been unknown: (a) Does PSA in human prostate cancer cells in tissue exist in a free or bound form? and (b) Is PSA produced by malignant cells in the free form because it has lost the ability to form a complex with ACT? Laser capture microdissection (LCM) enables the acquisition of pure populations of defined cell types from tissue (M. R. Emmert-Buck et al., Science, 274: 998-1001, 1996; R. F. Bonner et al., Science, 278: 1481-1483, 1997). This technology provides a unique opportunity to study intracellular protein composition and structure from human cells. In this study, we used LCM to assess the bound versus free form of intracellular PSA in both benign and malignant epithelium procured from prostate tissue. One-dimensional and two-dimensional PAGE were performed on cellular lysates from LCM-procured benign and malignant prostate epithelium from frozen tissue specimens. Western blotting analysis of one-dimensional PAGE gels revealed a strong band at M(r) 30,000 (expected molecular weight of unbound PSA) in all cases demonstrating that the vast majority of intracellular tumor and normal PSA exists within cells in the "free" form. Binding studies showed that PSA recovered from LCM-procured cells retained the full ability to bind ACT, and two-dimensional PAGE Western analysis demonstrated that the PSA/ACT complex was stable under strong reducing conditions. We conclude that intracellular PSA exists in the "free" form and that binding to ACT occurs exclusively outside of the cell.

Prevalence of microscopic tumors in normal appearing renal parenchyma of patients with hereditary papillary renal cancer.

PURPOSE: We describe the earliest renal lesions associated with hereditary papillary renal cancer and estimate the prevalence of microscopic papillary renal tumors. MATERIALS AND METHODS: Grossly normal tissue was obtained from 12 kidneys during renal surgery in 9 patients with hereditary papillary renal cancer. Tissue was examined microscopically and findings were compared to those previously reported to be associated with von Hippel-Lindau disease and sporadic renal cell carcinoma. RESULTS: A total of 92 microscopic papillary renal cell carcinoma lesions were identified on 46 of 88 slides (53%). No other lesions were identified. All tumors were solid and displayed the basophilic papillary histology characteristic of hereditary papillary renal cancer. Extrapolation of the data predicted the prevalence of 1,100 to 3,400 microscopic papillary tumors in a single kidney in a patient with hereditary papillary renal cancer. CONCLUSIONS: The basophilic papillary histology characteristic of clinically apparent renal tumors in patients with hereditary papillary renal cancer also characterizes the multiple microscopic lesions seen in the kidneys. These findings suggest that the earliest renal tumor in patients with an activating hereditary mutation of the met gene is papillary basophilic renal cancer. The large number of microscopic tumors in patients with hereditary papillary renal cancer was comparable to or greater than that seen in those with von Hippel-Lindau disease.

Prostate-specific antigen.

Serum prostate-specific antigen (PSA) measurements are the most useful serum biomarker to aid in early prostate cancer detection, clinical staging and therapeutic monitoring. Although the optimal use of PSA testing remains controversial, population based studies suggest that PSA screening reduces prostate cancer mortality. Customizing screening protocols based on individual risk factors and PSA level may be a useful approach to reduce overall costs incurred by widespread PSA testing. Lowering PSA cut-offs (i.e., from 4.0 ng/ml to 2.5 ng/ml) may reduce advanced stage prostate cancer, and the use of different PSA derivatives and PSA forms may reduce 'unnecessary' biopsies in some men. In addition to prostate cancer, manipulation and benign diseases of the prostate falsely elevate serum PSA levels. In contemporary clinical practice, PSA testing plays an important role in prostate cancer diagnosis and treatment.

Pain and morbidity of transrectal ultrasound guided prostate biopsy: a prospective randomized trial of 6 versus 12 cores.

PURPOSE: An increasing number of studies suggest that 6-sector transrectal ultrasound guided biopsy of the prostate provides insufficient material to detect all clinically important prostate cancers and more cores may improve detection rates. We performed a prospective, randomized study to determine the effect of increasing the number of cores from 6 to 12 on pain and other morbidity associated with the biopsy procedure. MATERIALS AND METHODS: A total of 160 men (44 black, 28%) with a mean age plus or minus standard deviation of 65+/-8 years who had serum prostate specific antigen between 2.5 and 20.0 ng./ml. and/or digital rectal examination findings suspicious for cancer were prospectively randomized to undergo 6 or 12-core biopsy. Patients completed a self-administered questionnaire addressing pain and other morbidity before, and immediately and 2 and 4 weeks after biopsy. RESULTS: There was no difference between groups in mean pain scale with time for abdominal and rectal pain. For probe insertion, needle insertion and overall pain there was a significant increase in pain recalled at 2 which persisted at 4 weeks compared to immediately after biopsy. However, there was no difference for these 3 post-biopsy pain measures between the 6 and 12-core groups. In the 12-core group there was a statistically significant increase in hematochezia and hematospermia (24% versus 10%, p = 0.04 and 89% versus 71%, p = 0.01, respectively) but no significant difference between groups reporting morbidity as a moderate or major problem. There was no significant change in International Prostate Symptom Score, fever or hospitalization in the 12-core group. CONCLUSIONS: The 12-core prostate biopsy procedure is generally well tolerated and can be safely performed with no significant difference in pain or morbidity compared to the 6-core procedure.

The age of the urologist affects the postoperative care of prostate carcinoma patients.

BACKGROUND: Strategies utilized by urologists in managing prostate carcinoma patients after radical prostatectomy vary appreciably. The reason for this is unclear. The authors investigated the effect of practitioner age on management strategies. METHODS: From among the total of 12,500 American Urological Association (AUA) members, 4467 were randomly selected to receive a custom-designed survey about their care of prostate carcinoma patients after radical prostatectomy. Respondents were asked to describe their follow-up practices for patients treated with curative intent, their motivations regarding postoperative surveillance, their methods of evaluating a postoperative increase in serum prostate specific antigen (PSA) level, and their choices of treatment for patients with recurrent prostate carcinoma. RESULTS: One thousand fifty responses were analyzed. There was a statistically significant influence of practitioner age on the management of at-risk patients, but it was quite small. The typical workup for an elevated postoperative serum PSA level also varied significantly according to practitioner age; older urologists ordered more serum prostatic acid phosphatase levels and computed tomography scans of the abdomen and pelvis, whereas younger urologists ordered more bone scans. The treatment of recurrent prostate carcinoma did not vary significantly according to urologist age. The opinions of older urologists regarding the survival benefits of postoperative surveillance were considerably different from the opinions of their younger colleagues. CONCLUSIONS: The results of this study suggest that urologist age accounts for some of the variation in the postoperative management of prostate carcinoma patients. Differences in beliefs regarding the benefits of surveillance may be partially responsible for this. Persuasive clinical research will probably be required to increase the uniformity of practice in this important area.

Anaemia in men receiving combined finasteride and flutamide therapy for advanced prostate cancer.

OBJECTIVE: To determine the effect of combined finasteride and flutamide therapy on haemoglobin and haematocrit values in men with advanced prostate cancer. PATIENTS AND METHODS: Nineteen men, previously untreated by hormone therapy, with histologically confirmed adenocarcinoma of the prostate and clinical evidence of advanced disease, were treated with combined finasteride (5 mg/day) and flutamide (750 mg/day) for at least 6 months. Complete blood counts were performed before initiation and after 6 months of therapy. RESULTS: After 6 months of finasteride and flutamide therapy both haemoglobin levels and haematocrit decreased in all men, with a mean (sd, range) decrease of 16 (10, 3-42) g/L and 4.6 (2.7, 0.8-9.9)%, respectively. CONCLUSION: Combined finasteride and flutamide therapy significantly lowers haemoglobin and haematocrit levels in men with advanced prostate cancer, and further study of this effect is warranted.

Current followup strategies after radical prostatectomy: a survey of American Urological Association urologists.

PURPOSE: Followup care of men who have undergone potentially curative surgical treatment for prostate cancer varies widely among clinicians. To determine current practice patterns we mailed a custom designed questionnaire to American and nonAmerican urologists who were American Urological Association (AUA) members. MATERIALS AND METHODS: Surveys were mailed to a random sample of the approximately 12,000 AUA members, comprising 3,205 Americans and 1,262 nonAmericans. Evaluable surveys were returned by 760 American (24%) and 290 nonAmerican (23%) urologists. Our analysis is based on these 1,050 responses. RESULTS: In generally healthy patients after radical prostatectomy for stages T1 to 2NOMO and T3a to cNOMO prostate cancer the most frequently recommended followup diagnostic tests included office visit with digital rectal examination, serum prostate specific antigen (PSA) and urinalysis. Although there is appreciable variation in the frequency of use of these methods, respondents generally recommended office visit with digital rectal examination, serum PSA and urinalysis every 3 months in year 1, every 6 months in years 2 to 5 and annually thereafter. Other tests, such as serum prostatic acid phosphatase, bone scan, and abdominal and pelvic computerized tomography and magnetic resonance imaging, are rarely recommended. CONCLUSIONS: Our survey provides information regarding current followup strategies recommended by AUA urologists after radical prostatectomy for stages T1 to 2NOMO and T3a to cNOMO disease. Office visits and digital rectal examination, urinalysis and PSA measurement are the main tools that urologists currently use. Although optimal strategy remains unknown, these data permit the rational design of clinical trials of alternate followup strategies based on actual current practice to answer this important question.

Evaluation and management of men whose radical prostatectomies failed: results of an international survey.

OBJECTIVES: To determine how urologists evaluate and treat men who develop recurrent prostate cancer after radical prostatectomy. METHODS: Surveys were mailed to 4467 American Urological Association members comprising 3205 U.S. and 1262 non-U.S. urologists randomly selected from a total membership of approximately 12,000. One thousand four hundred sixteen were returned and 1050 (760 U.S. and 290 non-U.S.) surveys were evaluable. RESULTS: To evaluate men with an elevated or rising prostate-specific antigen (PSA) level more than 1 year after radical prostatectomy, 98% of respondents use digital rectal examination, 68% use bone scan, 54% use transrectal ultrasound with biopsy, 36% use abdominal or pelvic computed tomography scan, 31% use transrectal ultrasound without biopsy, 25% use prostatic acid phosphatase, 11% use monoclonal antibody scan, and 5% use abdominal or pelvic magnetic resonance imaging. Respondents evaluate men with an elevated or rising PSA within 1 year of radical prostatectomy similarly. To treat documented local recurrence, 81% of respondents recommend radiation therapy, 7% recommend orchiectomy or luteinizing hormone-releasing hormone (LHRH) agonists, 6% recommend observation only, and 5% recommend combined androgen ablation. To treat documented distant recurrence, 50% recommend combined androgen ablation, 42% recommend orchiectomy or LHRH agonists, and 7% recommend observation only. To treat PSA-only recurrence, 54% recommend observation only, 16% recommend combined androgen ablation, 15% recommend orchiectomy or LHRH agonists, and 13% recommend radiation therapy. CONCLUSIONS: The evaluation of men whose radical prostatectomy failed varies among urologists and does not depend on time of recurrence. Radiation therapy is used by most urologists to treat local recurrence. Hormonal manipulation is used by more than 90% of urologists to treat distant recurrence. More than 50% of urologists recommend observation for men with biochemical-only recurrence.