Rainer Kuefer

Quantitative Determination of Expression of the Prostate Cancer Protein α-Methylacyl-CoA Racemase Using Automated Quantitative Analysis (AQUA) : A Novel Paradigm for Automated and Continuous Biomarker Measurements

Despite years of discovery and attempts at validation, few molecular biomarkers achieve acceptance in the clinical setting. Tissue-based markers evaluated by immunohistochemistry suffer from a high degree of inter- and intraobserver variability. One recent advance in this field that promises to automate this process is the development of AQUA, a molecular-based method of quantitative assessment of protein expression. This system integrates a set of algorithms that allows for the rapid, automated, continuous, and quantitative analysis of tissue samples, including the separation of tumor from stromal elements and the subcellular localization of signals. This study uses the AQUA system to assess a recently described prostate cancer biomarker, alpha-methylacyl-CoA-racemase (AMACR), and to determine the effectiveness of the quantitative measurement of this marker as a means for making the diagnosis of prostate cancer. Using a prostate cancer progression tissue microarray containing a wide range of prostate tissues, AQUA was directly compared to standard immunohistochemical evaluation for AMACR protein expression using the p504s monoclonal antibody. Both methods produced similar results showing AMACR protein expression to be strongest in the clinically localized prostate cancer, followed by the metastatic tumor samples. Benign prostate tissue was categorized as negative for most tissue samples by immunohistochemistry. However, AMACR was detectable using the AQUA system at low levels using the standard 1:25 dilution but also at 1:250 dilution, which is not detectable by light microscopy. The AQUA system was also able to discriminate foamy gland prostate cancers, which are known to have a lower AMACR expression than typical acinar prostate cancers, from benign prostate tissue samples. Finally, a receiver-operating-characteristic curve was plotted to determine the specificity of the AMACR AQUA Z-score (normalized AQUA score) to predict that a given tissue microarray sample contains cancer. The area under the curve was calculated at 0.90 (P < 0.00001; 95% CI, 0.84 to 0.95). At an AMACR AQUA Z-score score of -0.3, 91% of the 70 samples classified as prostate cancer were correctly categorized without the intervention of a pathologist reviewing the tissue microarray slide. In conclusion, the AQUA system provides a continuous measurement of AMACR on a wide range of prostate tissue samples. In the future, the AMACR AQUA Z-score may be useful in the automated screening and evaluation of prostate tissue biomarkers.

Treatment-Dependent Androgen Receptor Mutations in Prostate Cancer Exploit Multiple Mechanisms to Evade Therapy

Mutations in the androgen receptor (AR) that enable activation by antiandrogens occur in hormone-refractory prostate cancer, suggesting that mutant ARs are selected by treatment. To validate this hypothesis, we compared AR variants in metastases obtained by rapid autopsy of patients treated with flutamide or bicalutamide, or by excision of lymph node metastases from hormone-naïve patients. AR mutations occurred at low levels in all specimens, reflecting genetic heterogeneity of prostate cancer. Base changes recurring in multiple samples or multiple times per sample were considered putative selected mutations. Of 26 recurring missense mutations, most in the NH(2)-terminal domain (NTD) occurred in multiple tumors, whereas those in the ligand binding domain (LBD) were case specific. Hormone-naïve tumors had few recurring mutations and none in the LBD. Several AR variants were assessed for mechanisms that might underlie treatment resistance. Selection was evident for the promiscuous receptor AR-V716M, which dominated three metastases from one flutamide-treated patient. For the inactive cytoplasmically restricted splice variant AR23, coexpression with AR enhanced ligand response, supporting a decoy function. A novel NTD mutation, W435L, in a motif involved in intramolecular interaction influenced promoter-selective, cell-dependent transactivation. AR-E255K, mutated in a domain that interacts with an E3 ubiquitin ligase, led to increased protein stability and nuclear localization in the absence of ligand. Thus, treatment with antiandrogens selects for gain-of-function AR mutations with altered stability, promoter preference, or ligand specificity. These processes reveal multiple targets for effective therapies regardless of AR mutation.

Overexpression, Amplification, and Androgen Regulation of TPD52 in Prostate Cancer

Gains in the long arm of chromosome 8 (8q) are believed to be associated with poor outcome and the development of hormone-refractory prostate cancer. Based on a meta-analysis of gene expression microarray data from multiple prostate cancer studies (D. R. Rhodes et al., Cancer Res 2002;62:4427–33), a candidate oncogene, Tumor Protein D52 (TPD52), was identified in the 8q21 amplicon. TPD52 is a coiled-coil motif-bearing protein, potentially involved in vesicle trafficking. Both mRNA and protein levels of TPD52 were highly elevated in prostate cancer tissues. Array comparative genomic hybridization and amplification analysis using single nucleotide polymorphism arrays demonstrated increased DNA copy number in the region encompassing TPD52. Fluorescence in situ hybridization on tissue microarrays confirmed TPD52 amplification in prostate cancer epithelia. Furthermore, our studies suggest that TPD52 protein levels may be regulated by androgens, consistent with the presence of androgen response elements in the upstream promoter of TPD52. In summary, these findings suggest that dysregulation of TPD52 by genomic amplification and androgen induction may play a role in prostate cancer progression.

Multiple Novel Prostate Cancer Predisposition Loci Confirmed by an International Study: The PRACTICAL Consortium

A recent genome-wide association study found that genetic variants on chromosomes 3, 6, 7, 10, 11, 19 and X were associated with prostate cancer risk. We evaluated the most significant single-nucleotide polymorphisms (SNP) in these loci using a worldwide consortium of 13 groups (PRACTICAL). Blood DNA from 7,370 prostate cancer cases and 5,742 male controls was analyzed by genotyping assays. Odds ratios (OR) associated with each genotype were estimated using unconditional logistic regression. Six of the seven SNPs showed clear evidence of association with prostate cancer (P = 0.0007-P = 10−17). For each of these six SNPs, the estimated per-allele OR was similar to those previously reported and ranged from 1.12 to 1.29. One SNP on 3p12 (rs2660753) showed a weaker association than previously reported [per-allele OR, 1.08 (95% confidence interval, 1.00-1.16; P = 0.06) versus 1.18 (95% confidence interval, 1.06-1.31)]. The combined risks associated with each pair of SNPs were consistent with a multiplicative risk model. Under this model, and in combination with previously reported SNPs on 8q and 17q, these loci explain 16% of the familial risk of the disease, and men in the top 10% of the risk distribution have a 2.1-fold increased risk relative to general population rates. This study provides strong confirmation of these susceptibility loci in multiple populations and shows that they make an important contribution to prostate cancer risk prediction. (Cancer Epidemiol Biomarkers Prev 2008;17(8):2052–61)

Evaluation of [11C]‐choline positron‐emission/computed tomography in patients with increasing prostate‐specific antigen levels after primary treatment for prostate cancer

To evaluate [11C]‐choline positron‐emission tomography (PET)/computed tomography (CT) for detecting clinical recurrence after primary treatment for prostate cancer.

α-Methylacyl-CoA Racemase: Expression Levels of this Novel Cancer Biomarker Depend on Tumor Differentiation

alpha-Methylacyl-CoA racemase (AMACR) has previously been shown to be a highly sensitive marker for colorectal and clinically localized prostate cancer (PCa). However, AMACR expression was down-regulated at the transcript and protein level in hormone-refractory metastatic PCa, suggesting a hormone-dependent expression of AMACR. To further explore the hypothesis that AMACR is hormone regulated and plays a role in PCa progression AMACR protein expression was characterized in a broad range of PCa samples treated with variable amounts and lengths of exogenous anti-androgens. Analysis included standard slides and high-density tissue microarrays. AMACR protein expression was significantly increased in localized hormone-naive PCa as compared to benign (P < 0.001). Mean AMACR expression was lower in tissue samples from patients who had received neoadjuvant hormone treatment but still higher compared to hormone-refractory metastases. The hormone-sensitive tumor cell line, LNCaP, demonstrated stronger AMACR expression by Western blot analysis than the poorly differentiated cell lines DU-145 and PC-3. AMACR protein expression in cells after exposure to anti-androgen treatment was unchanged, whereas prostate-specific antigen, known to be androgen-regulated, demonstrated decreased protein expression. Surprisingly, this data suggests that AMACR expression is not regulated by androgens. Examination of colorectal cancer, which is not hormone regulated, demonstrated high levels of AMACR expression in well to moderately differentiated tumors and weak expression in anaplastic colorectal cancers. Taken together, these data suggest that AMACR expression is not hormone-dependent but may in fact be a marker of tumor differentiation.

The Role of Metastasis-Associated Protein 1 in Prostate Cancer Progression

Distinguishing aggressive prostate cancer from indolent disease represents an important clinical challenge, as current therapy requires over treating men with prostate cancer to prevent the progression of a few cases. Expression of the metastasis-associated protein 1 (MTA1) has previously been found to be associated with progression to the metastatic state in various cancers. Analyzing DNA microarray data, we found MTA1 to be selectively overexpressed in metastatic prostate cancer compared with clinically localized prostate cancer and benign prostate tissue. These results were validated by demonstrating overexpression of MTA1 in metastatic prostate cancer by immunoblot analysis. MTA1 protein expression was evaluated by immunohistochemistry in a broad spectrum of prostate tumors with tissue microarrays containing 1940 tissue cores from 300 cases. Metastatic prostate cancer demonstrated significantly higher mean MTA1 protein expression intensity (score = 3.4/4) and percentage of tissue cores staining positive for MTA1 (83%) compared with clinically localized prostate cancer (score = 2.8/4, 63% positive cores) or benign prostate tissue (score = 1.5/4, 25% positive cores) with a mean difference of 0.54 and 1.84, respectively (P < 0.00001 for both). Paradoxically, for localized disease, higher MTA1 protein expression was associated with lower rates of prostate specific antigen recurrence after radical prostatectomy for localized disease. In summary, this study identified an association of MTA1 expression and prostate cancer progression.

Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer

Histone deacetylase inhibitors (HDACs) are known to exhibit antiproliferative effects on various carcinoma cells. In this study, the in vivo efficiency of two HDACs, sodium butyrate and tributyrin, on prostate cancer growth inhibition were investigated. To gain an insight into the possible underlying pathways, cell culture experiments were performed focusing on the expression of p21, Rb and c-myc. For in vivo testing, prostate cancer cell lines (PC3 and TSU-Pr1) were seeded on the chorioallantois membrane (CAM) and implanted in a xenograft model using nude mice. Standard Western blot analysis was performed for protein expression of p21, Rb and c-myc in HDAC-treated vs untreated prostate cancer cells. Both sodium butyrate and tributyrin had a considerable treatment effect on microtumours on the chicken egg at already very low concentrations of 0.1 mM. Tributyrin-treated tumours showed the strongest effect with 38% apoptotic nuclei in the prostate cancer cell line PC3. In the mouse model, there was almost no difference between sodium butyrate and tributyrin. In untreated animals the tumours were almost double the size 4 weeks after implantation. Tumours of the treatment groups had a significantly lower percentage of Ki-67-positive-stained nuclei. As demonstrated by Western blot analysis, these effects seem to be independent of p53 status and a pathway via p21–Rb–c-myc is possibly involved. In this study we have demonstrated a substantial in vivo treatment effect, which can be induced by the application of sodium butyrate or the orally applicable tributyrin in human prostate cancer. The given results may provide the rationale to apply these drugs in well-controlled clinical trials in patients being at high risk of recurrence after specific therapy or in patients with locally or distant advanced prostate cancer.

Oncological Followup After Radical Cystectomy for Bladder Cancer—Is There Any Benefit?

PURPOSE Tumor recurrence after radical cystectomy for bladder cancer can be detected in an asymptomatic patient by regular followup or in a symptomatic patient by symptom guided examination. To our knowledge it is still unknown whether detecting tumor recurrence at an asymptomatic stage offers a better survival rate. MATERIALS AND METHODS A total of 1,270 radical cystectomies for bladder cancer were performed at a single institution between January 1, 1986 and December 2006. All patients had regular followup examinations with chest x-ray and abdominal ultrasound every 3 months, computerized tomography of the abdomen every 6 months, and bone scan and excretory urography every 12 months. Additional examinations were required for symptomatic disease. We analyzed the first site and date of tumor recurrence. Survival was compared using the log rank test. RESULTS The 20-year recurrence rate was 48.6% in the complete series. Tumor recurrence developed in 444 patients, including 154 asymptomatic and 290 symptomatic patients, with a mean time after radical cystectomy of 20 and 17.5 months, respectively. The most frequent symptoms were pain, ileus, acute urinary retention, hydronephrosis with flank pain, hematuria, neurological symptoms and a palpable mass. Of the 444 patients 182 (41%) had local recurrence and 324 (73%) had distant failure at the time of first recurrence. The overall survival rate 1, 2 and 5 years after first recurrence was 22.5%, 10.1% and 5.5% in asymptomatic patients, and 18.9%, 8.2% and 2.9% in symptomatic patients, respectively (log rank not significant). CONCLUSIONS This study fails to demonstrate a survival benefit for detecting tumor recurrence early at an asymptomatic stage by regular followup examinations. These data show that symptom guided followup examinations may provide similar results at lower cost.

ERG rearrangement is specific to prostate cancer and does not occur in any other common tumor

Identification of specific somatic gene alterations is crucial for the insight into the development, progression, and clinical behavior of individual cancer types. The recently discovered recurrent ERG rearrangement in prostate cancer might represent a prostate cancer-specific alteration that has not been systematically assessed in tumors other than prostate cancer. Aim of this study was to assess, whether the ERG rearrangement and the distinct deletion site between TMPRSS2 and ERG, both predominantly resulting in a TMPRSS2–ERG fusion, occur in tumors other than prostate cancer. We assessed 54 different tumor types (2942 samples in total) for their ERG rearrangement status by fluorescence in situ hybridization (FISH). To calibrate, we analyzed 285 prostate cancer samples for the ERG rearrangement frequency. Additionally, we interrogated a high-resolution single nucleotide polymorphism (SNP) data set across 3131 cancer specimens (26 tumor types) for copy number alterations. None of the 54 different tumor types assessed by FISH harbored an ERG rearrangement, whereas the prostate cancer samples revealed an ERG rearrangement in 49.5% of cases. Furthermore, within the 26 tumor types assessed for copy number alterations by SNP, the distinct deletion site between TMPRSS2 and ERG (21q22.2–3) was detectable exclusively in prostate cancer. Although Ewings sarcoma and AML have known rearrangements rarely involving ERG, we hypothesize that the ERG rearrangement as well as the distinct deletion site on 21q22.2–3 between TMPRSS2 and ERG are prostate-cancer-specific genomic alterations. These observations provide further insight into the oncogenesis of prostate cancer and might be critical for the development of ERG rearrangement assessment as a clinical tool.