The continuous debate on whether current serum prostate-specific antigen (PSA)–based screening strategies are potentially leading to “overtreatment” of a subset of patients with prostate cancer (PCa) has further fueled the interest in pursuing clinicopathologic and molecular parameters that may help identify patients with biologically “significant” prostate cancers.162,163
A parallel pursuit of clinicopathologic algorithms and criteria that can accurately predict “insignificant” PCa tumors is also gaining momentum. The latter are generally defined as tumors that lack the biologic potential to affect disease-specific mortality and morbidity within a given patient life expectancy. As alternative PCa management approaches, such as “proactive surveillance,” are increasingly offered, accurate identification of insignificant PCa becomes more pressing.
Meanwhile, prostate needle biopsy continues to be the gold standard for establishing the diagnosis of PCa in patients with elevated serum PSA levels and/or positive digital rectal examination results. Established clinicopathologic parameters including clinical stage, pathologic stage, histologic Gleason grade, and serum PSA levels are the sole guiding tools of prognostication and disease management in PCa.164–166
Given the existing need to improve upon the prognostic and predictive power of the above-established parameters, an extensive list of molecular biomarkers have been evaluated in the last decade for their potential role in enhancing our ability to predict disease progression, response to therapy, and survival.167–171
These research efforts have been greatly facilitated by the wealth of information garnered from gene expression array studies and by sophisticated bioinformatics tools evaluating the overwhelming data sets generated from genomic, transcriptomic, and proteomic studies. These genomic technologies continue to yield new markers that can in turn be evaluated for clinical utility in a high-throughput manner with IHC and FISH-labeled tissue microarrays and state-of-the art image analysis systems.172–174
In the last decade, steps detailing the many genetic alterations involved in the progression of PCa have been unveiled (). The discovery by Tomlins et al175,176
of a recurrent chromosomal rearrangement in more than one-half of their analyzed PCa cases is ranked as one of the most notable in solid tumor biology, given the shear prevalence of PCa. The recurrent chromosomal rearrangements lead to a fusion of the androgen-responsive promoter elements of the TMPRSS2
gene (21q22) to 1 of 3 members of the ETS
transcription factor family members–ERG, ETV1,
–located at chromosome bands 21q22, 7p21, and 17q21, respectively. Although the prognostic role of assessing TMPRSS2-ETS
rearrangements in PCa tissue samples has been called into question by recent, well-designed large cohort studies,177,178
the discovery will no doubt have great implications in terms of furthering our understanding of the steps involved in the development and pathogenesis of PCa and will provide a new marker for molecular diagnosis and potential target(s) of therapy in PCa.179–188
The potential diagnostic and prognostic role of detecting TMPRSS2-ERG
in post–prostate massage urine samples requires further investigation.189–191
depicts a commonly used FISH split apart–based approach for the evaluation of ERG
Molecular alterations involved in oncogenesis and progression of prostate cancer. Abbreviations: PCa, prostate adenocarcinoma; PIN, prostatic intraepithelial neoplasia; PTEN, phosphatase and tensin homolog.
Figure 4 Fluorescence in situ hybridization–based evaluation of ERG gene fusion in prostate carcinoma. A, A normal ERG allele will hybridize with the ERG 3′ and the ERG 5′ probes, leading to the formation of juxtaposed green-red signals (more ...)
Recently, commercial anti-ERG monoclonal antibodies became available that make it possible to use IHC for evaluating ERG protein expression as a surrogate approach to detecting TMPRSS2-ERG
fusion by FISH. We, and others, have demonstrated a strong correlation between ERG overexpression by IHC and ERG
fusion status with rates greater than 86% for sensitivity and specificity (see and ). Immunohistochemistry may offer an accurate, simpler, and less costly alternative for evaluation of ERG fusion status in PCa on needle biopsy and radical prostatectomy samples.192,193
Immunohistochemistry Expression of ERG Protein Strongly Correlates With TMRSS2-ERG Fusion Regardless of Mechanism of Fusiona
Figure 5 Overexpression of ERG, as demonstrated by immunohistochemistry, is a simple surrogate method for evaluating TMPRSS2-ERG fusion in prostate adenocarcinoma. Positive expression of ERG in Gleason grades 6 and 8 cases that were also positive for TMPRSS2-ERG (more ...)
The perceived need to identify “objective” markers to supplement, or conceivably supplant, the more “subjective” established histologic parameters has been a major driving force behind biomarker discovery efforts. It is crucial to recognize and account for the potential variability that can exist even with the new molecular parameters. Sources of variability include differences in molecular technique methodologies, tissue fixation and processing, interobserver and intraobserver variability (in IHC-based biomarkers), and differences in cutoff points.194
Furthermore, illustration of statistical significance for a particular biomarker does not alone assure its utility for a given patient. Therefore, a promising prognostic or therapeutic target biomarker should endure a rigorous “evidence-based” analysis and be validated in large, prospective clinical trials before transition into standard practice.195
Emerging Prognostic Factors
Currently pursued prognostic molecular biologic markers in PCa are categorized as College of American Pathologists Consensus Category III Prognostic Factors.196
Category III prognostic factors are those still needing additional studies to assure their prognostic utility before undergoing further clinical trials. In contrast, Category I factors are considered as proven to be useful in clinical practice and include preoperative PSA, TNM stage, Gleason grade, and surgical margins. Category II factors are factors that have been extensively studied but await statistically robust trials and include parameters such as tumor volume, histologic type, and DNA ploidy analysis.
The wide array of molecular–based PCa markers include proliferation index (Ki-67), microvessel density, nuclear morphometry, tumor suppression genes (eg, p53, p21, p27, NKX3.1, phosphatase and tensin homolog [PTEN], and retinoblastoma gene [Rb]), oncogenes (eg, Bcl2, c-myc, EZH2, and HER2), adhesion molecules (CD44, E-cadherin), PI3K/akt/mTOR pathway,197,198
apoptosis regulators (eg, survivin and transforming growth factor β 1), androgen receptor status, neuroendocrine differentiation markers, and prostate tissue lineage-specific marker expression (PSA, prostate-specific alkaline phosphatase, and prostate-specific membrane antigen).199–201
A single study202
has so far found proliferation index, as measured by Ki-67 and percentage of cells in S phase and G2M, to be superior to Gleason score in predicting biochemical recurrence after radical prostatectomy. Two additional studies203,204
have shown a similar role for Ki-67 index measurement as an independent prognosticator in prostatectomy specimens. Conflicting reports have been furthered by others.205–208
The mean number of microscopic blood vessels in tissue is higher in PCa and prostatic intraepithelial neoplasia than normal prostate tissue. In a study evaluating microvessel density (MVD) on needle biopsy, the authors209
found that MVD, when combined with Gleason score and preoperative PSA, provided improved ability to predict extraprostatic extension at radical prostatectomy. Although MVD was significant in the multivariate analysis, Gleason score and serum PSA levels were much more powerful predictors of extraprostatic disease. Three additional studies210–212
revealed a prognostic role for MVD in prostatectomy specimens. Others,213–215
however, failed to confirm such a role. Differences in vascular antibodies used and topography of vessel measurements could account for the variable results. It appears that MVD will have a marginal adjunctive role, if any, to established current parameters.
Tumor Suppressor Genes and Oncogenes
Among tumor suppressor genes, there is mounting evidence to support a role for p53 expression in predicting prognosis in PCa. Brewster et al found216
p53 expression and Gleason score in needle biopsy to be independent predictors of biochemical relapse after radical prostatectomy. Another study217
found p53 status on prostatectomy but not needle biopsies to be predictive, raising the issue of sampling. Many studies evaluating prostatectomy specimens found p53 to be of prognostic significance, independent of grade, stage, and margin status.207,215,218–223
The results of these studies suggest that p53 evaluation could become a clinically used parameter, at least in prostatectomy specimens, once standardization of cutoffs and immunostaining methodologies are achieved in large prospective studies. Most studies of another tumor suppressor gene, p27, a cell cycle inhibitor, have also supported a correlation with progression after prostatectomy.208,224
Several recent studies have demonstrated that the PTEN/PI3K/mTOR pathway plays an important role in cell growth, proliferation, and oncogenesis in prostate cancer.225–231
Phosphatase and tensin homolog is a negative regulator of this pathway. Loss of PTEN tumor suppressor gene activity and the ensuing mTOR pathway activation appear to be associated with poor prognosis in prostate cancer. The mTOR pathway is also a potential target for prostate cancer treatment, and several rapamycin analogs are currently being tested as potential therapeutic agents for PCa.197,230
We recently reported the results of a pilot study evaluating the pharmacodynamic efficacy of neoadjuvant rapamycin therapy in PCa.197
Using IHC analysis, we found a significant decrease in Phos-S6 protein, the main downstream effector of mTOR pathway, in patients receiving neoadjuvant therapy.197
While less robust evidence exists for the prognostic role of p21,232
a downstream mediator of p53, and transcription factors such as NKX3.1,172,233
preponderance of evidence supports a prognostic role for Bcl2203,216,218,220,222
and myc oncogenes234,235
as potential adjuncts to histologic prognostic parameters.
Despite great interest in HER2 and its potential use as a target of therapy, the data on its relation to prognosis in PCa are conflicting, with 1 study by Veltri et al236
showing HER2 to be an independent prognosticator and more recent studies using both IHC and FISH assessment showing lack of its utility in predicting progression.237
In an elegant gene expression profiling study using cDNA microarrays containing 26 000 genes, Lapointe et al238
identified 3 subclasses of prostate tumors by distinct patterns of gene expression. High-grade and advanced-stage tumors, as well as tumors associated with recurrence, were disproportionately represented among 2 of the 3 subtypes, one of which also included mostly lymph node metastases. Furthermore, 2 surrogate genes were differentially expressed among tumor subgroups by IHC. These included MUC1, a gene highly expressed in the subgroups with “aggressive” clinicopathologic features, and AZGP1, a gene highly expressed in the favorable subgroup. The 2 surrogate markers were strong predictors of tumor recurrence, independent of tumor grade, stage, and preoperative PSA levels. Such study suggests that prostate tumors can be classified according to their gene expression patterns; these tumor subtypes may provide a basis for improved prognostication and treatment stratification.
In another study, Tomlins et al239
used laser-capture microdissection to isolate 101 cell populations to illustrate gene expression profiles of PCa progression from benign epithelium to metastatic disease. By analyzing expression signatures in the context of more than 14 000 “molecular concepts,” or sets of biologically connected genes, the authors generated an integrative model of progression. Molecular critical transitions in progression included protein biosynthesis, E26 transformation-specific (ETS) family transcriptional targets, androgen signaling, and cell proliferation. Known prognostic markers, such as grade, could be ascribed to noted attenuated androgen-signaling signature seen in high-grade cancer (Gleason pattern 4), similar to metastatic prostate cancer, which may reflect dedifferentiation and explain the clinical association of grade and prognosis. Taken together, these data show that analyzing gene expression signatures in the context of a compendium of molecular concepts is useful in understanding cancer biology.
Lapointe et al240
complemented their above-mentioned gene expression findings by looking for associated copy number alterations with array-based comparative genomic hybridization. They were able to identify recurrent copy number genetic aberrations240
corresponding to 3 prognostically distinct groups of PCa: (1) deletions at 5q21 and 6q15 deletion group, associated with favorable outcome; (2) a 8p21 (NKX3-1) and 21q22 (resulting in TMPRSS2-ERG fusion) deletion group, and (3) gains in 8q24 (MYC) and 16p13, and loss at 10q23 (PTEN) and 16q23 groups, correlating with metastatic disease and aggressive outcome.
Finally, in a recent genome-wide analysis of PCa, Taylor et al241
elegantly illustrated how detailed annotation of PCa genomes can affect our understanding of the disease and its treatment strategy. Assessing DNA copy number, messenger RNA expression, and focused exon resequencing in 218 prostate cancer tumors, the authors identified the role of nuclear receptor coactivator NCOA2 as a novel oncogene in 11% of PCa cases. TMPRSS2-ERG fusion was associated with novel prostate-specific deletion at chromosome band 3p14, which may implicate FOXP1, RYBP, and SHQ1 as potential cooperative tumor suppressors. Most intriguing was their ability to define clusters of lowrisk and high-risk disease beyond that achieved by Gleason score by using DNA copy number data. As shown in , six clusters of PCa tumors are identified by unsupervised hierarchical clustering with distinct risk for biochemical recurrence.
Figure 6 Clinically distinct groups of prostate cancer are identified by genomic alterations. A, Unsupervised hierarchical clustering of copy number alterations identified 6 groups (clusters) of prostate cancers. B, Statistically significant differences in freedom (more ...)
Genomic studies suggest that prostate cancers develop via a limited number of alternative preferred genetic pathways. The resultant molecular genetic subtypes provide a new framework for investigating PCa biology and explain, in part, the clinical heterogeneity of the disease.
Emerging Early Detection Markers and Targets of Therapy
Markers of PCa detection that can be applied to blood, urine, or prostatic secretion fluid (ejaculate or prostate massage fluids) are of great interest and have been the focus of active research. Markers that have been investigated in the urine or prostatic secretions include gene promoter hypermethylation profile assays242–245
and differential display code 3 (DD3), also known as PCA3 (). DD3 is a gene that expresses a noncoding RNA and was initially identified by Bussemakers et al246
as one of the most specific markers of PCa. Quantitative real-time reverse transcriptase PCR assay detecting PCA3 can be applied to blood, urine, or prostatic fluid.247
Figure 7 Structure of the PCA3/DD3 gene. The gene, which expresses a noncoding RNA, was mapped to chromosome 9q21–22 and consists of 4 exons. Alternative polyadenylation at 3 different positions in exon 4 (indicated as 4a, 4b, and 4c) gives rise to 3 different-sized (more ...)
Evaluation of PCA3 in postattentive prostate massage urine samples with transcription-mediated amplification technology has shown to be superior to serum PSA determination in predicting biopsy outcome, with sensitivity and specificity approximating 70% and 80%, respectively, and a negative predictive value of 90%248–251
; it is currently under evaluation for FDA approval in the United States. Encouraging data from the REDUCE trial support a role for evaluation of PCA3 in postattentive prostate massage urine sample in predicting positive prostate needle biopsy findings in immediately subsequent, as well as future, biopsies after an initial negative biopsy result. PCA3 may also have a role in predicting the risk for higher Gleason score and larger tumor volume on radical retropubic prostatectomy. If confirmed, the latter could be of great value in treatment option algorithms and in delineation of candidates for active surveillance.252–255
Multiplex urine assays to include PCA3, TMPRSS-ERG, SPINK1, and GOLPH2 are also under evaluation, with recent data suggesting an improved performance of such assays compared to PCA3 alone.256
Finally, several markers are being investigated as potential targets of therapy for prostate cancer. The list includes tyrosine kinase receptors (eg, EGFR), angiogenesis targets (eg, VEGF),257
fatty acid synthase,258
and prostate-specific membrane antigen,262–265
to name a few.