Date Published: September 13, 2012
Publisher: Public Library of Science
Author(s): Roble G. Bedolla, Jingjing Gong, Thomas J. Prihoda, I-Tien Yeh, Ian M. Thompson, Rita Ghosh, Addanki P. Kumar, Chad Creighton. http://doi.org/10.1371/journal.pone.0044917
Prediction of prostate cancer prognosis is challenging and predictive biomarkers of recurrence remain elusive. Although prostate specific antigen (PSA) has high sensitivity (90%) at a PSA level of 4.0 ng/mL, its low specificity leads to many false positive results and considerable overtreatment of patients and its performance at lower ranges is poor. Given the histopathological and molecular heterogeneity of prostate cancer, we propose that a panel of markers will be a better tool than a single marker. We tested a panel of markers composed of the anti-apoptotic protein FLIP and its transcriptional regulators Sp1 and Sp3 using prostate tissues from 64 patients with recurrent and non-recurrent cancer who underwent radical prostatectomy as primary treatment for prostate cancer and were followed with PSA measurements for at least 5 years. Immunohistochemical staining for Sp1, Sp3, and FLIP was performed on these tissues and scored based on the proportion and intensity of staining. The predictive value of the FLIP/Sp1/Sp3 signature for clinical outcome (recurrence vs. non-recurrence) was explored with logistic regression, and combinations of FLIP/Sp1/Sp3 and Gleason score were analyzed with a stepwise (backward and forward) logistic model. The discrimination of the markers was identified by sensitivity-specificity analysis and the diagnostic value of FLIP/Sp1/Sp3 was determined using area under the curve (AUC) for receiver operator characteristic curves. The AUCs for FLIP, Sp1, Sp3, and Gleason score for predicting PSA failure and non-failure were 0.71, 0.66, 0.68, and 0.76, respectively. However, this increased to 0.93 when combined. Thus, the “biomarker signature” of FLIP/Sp1/Sp3 combined with Gleason score predicted disease recurrence and stratified patients who are likely to benefit from more aggressive treatment.
Prostate cancer (PCA) is the second leading cause of cancer-related death in men and is expected to cause 28,170 deaths in the United States in 2012 . PCA generally affects men over 65 years of age but remains indolent and asymptomatic in a majority of cases. The histopathological and molecular heterogeneity of the disease makes prediction of prognosis challenging. Although PSA is the most widely used serum marker for prostate cancer, it has no accepted cut-off point with high sensitivity and specificity and often leads to false positive results –. Furthermore, there are currently no molecular markers that can be used to reliably predict which premalignant lesions will recur or develop into invasive PCA –. A valid biomarker should have the following characteristics: (i) accuracy (should not falsely predict positive or negative results); (ii) selectivity (ability to diagnose the disease during disease progression); and (iii) specificity (ability to distinguish cancerous from non-cancerous phenotype). Although PSA fulfills most of these criteria and is widely used, it is limited by its low values of specificity and selectivity –. Because of the growing evidence for overtreatment of prostate cancer, it is important to identify and validate new prognostic markers that will predict clinically significant prostate cancer –. Such markers will enable the targeted treatment of patients with aggressive tumors while avoiding unnecessary treatment and its side effects in patients with indolent disease.
In this study we assessed the expression of the anti-apoptotic protein FLIP and its transcription regulators Sp1 and Sp3 by immunohistochemical evaluation of tissue samples obtained from 64 patients who underwent radical prostatectomy as primary treatment for prostate cancer. Patients had at least 60 months follow-up with PSA measurements and only those with an undetectable PSA at 60 months were considered to have non-recurrent disease. Increasing levels of PSA after prostatectomy were used as a surrogate endpoint for poor outcome. PSA non-failure was defined as PSA levels undetectable or <0.2 ng/mL for at least 5 years after prostatectomy and no other signs of recurrence such as metastasis. PSA failure was defined as a PSA level >0.2 ng/mL that increased during the 5 years after prostatectomy . Due to limited sample size only two-way interactions were considered and PSA was not added to the Gleason score. First, we compared the expression of FLIP, Sp1, and Sp3 between the two groups using immunohistochemistry and found significant differences between PSA failure and non-failure groups in the expression of FLIP, Sp3, and Sp1 (Wilcoxon rank-sum; Figure 1 and Figure 2). As shown in the box plots in Figure 1, we found significant differences in the mean total IHC score between the non-recurrent and recurrent cases for Sp1 (p = 0.019), Sp3 (p = 0.011), and FLIP (p = 0.0019). We also included Gleason score in our analysis because this will have an influence on the outcome. Gleason scores for our 64-patient cohort were significantly different in the recurrent and non-recurrent groups (p = 0.0001; data not shown). It should be mentioned that this is not necessarily the case as studies have shown that Gleason grade 7 by itself may not be significant . In our cohort, 50% of prostatectomy cases were Gleason 7: (29.69% were 3+4 and 20.3% were 4+3). Of the 29.69% that were 3+4, 41.2% were non-recurrent and 16.67% were recurrent cases. On the other hand, of the 20.3% with the more aggressive 4+3 grading, 8.8% were non-recurrent and 33.33% were recurrent. These data suggest that the differences in FLIP, Sp1, and Sp3 between the biochemically recurrent and non-recurrent groups are significant.
Effective clinical management of PCA has been hampered by significant intratumoral heterogeneity combined with an incomplete understanding of the molecular events associated with the development of the disease and subsequent recurrence following traditional treatments –. Therefore, there is an unmet need for new methods and/or agents for PCA management. Given the individual genetic variation and the heterogeneity of the disease, personalized treatment approaches are critical for successful management of PCA. To develop such individualized treatment approaches, it is essential to identify a panel of biomarkers or a “biomarker signature” that could be used to stratify patients according to response to specific treatments –. Although serum-based PSA screening is widely used, PSA has the following limitations as an early detection biomarker –: (i) Elevated levels of serum PSA have been observed not only in prostate cancer, but also in benign prostatic hyperplasia patients, therefore PSA is not specific to prostate cancer, and (ii) PSA is not sufficiently sensitive as indicated by the Prostate Cancer Prevention Trial (PCPT), which demonstrated that 15% of men with PSA levels of 4 ng/ml had prostate cancer and 15% of these patients had high Gleason grade disease. In addition, two randomized trials showed a modest effect of PSA screening on prostate cancer mortality, suggesting a substantial risk of negative biopsy and overdiagnosis and overtreatment of indolent cancer. Although numerous markers including α-methyacylCoA-racemase (AMCAR), fatty acid synthetase (FASN), ERG, and prostate-specific membrane antigen (PSMA), have been identified based on preclinical studies and shown to be associated with the outcome of prostate cancer after surgical treatment using human tissue samples, very few of these have predictive value independent of traditional prognostic factors such as Gleason score, pathological stage, and pretreatment PSA levels –. To the best of our knowledge, there are currently no sensitive markers to monitor disease recurrence.