The RTOG can be credited with having performed the most extensive studies of biomarkers in men with clinically localized prostate cancer treated with external beam radiotherapy (EBRT) on Phase III Randomized Trials. A partial list of these studies is shown in . The source of the tissue used for these studies came from two Phase III Trials RTOG 8610 and RTOG 9202. Altogether eleven markers have been studied in 15 manuscripts between 1997 and 2008.
Selected Series of Biomarker studies performed by the RTOG
The first biomarker evaluated by the RTOG was p53. The study population consisted of a subset men entered on RTOG protocol 8610, treated with EBRT with or without combined androgen deprivation therapy (ADT) 4
. This subset consisted of 129 (27%) of the 471 patients entered in the trial, for whom there was sufficient tumor material for analysis. Abnormal p53 protein expression was detected in 23 (18%) of tumors. Statistically significant associations were noted between abnormal p53 protein expression and increased risk of distant metastases (P=0.04), decreased probability of progression-free survival (p=0.03), and reduction in overall survival (p=0.02). Patients treated with EBRT and ADT, whose tumors exhibited abnormal p53 protein expression had a reduced time to the development of metastases (P = 0.001), however this difference was not seen patients treated with EBRT alone. Based on this preliminary analysis, the RTOG carefully evaluated this endpoint for patients treated on RTOG 9202.
Tumor tissue sufficient for analysis of p53 status was available in 777 cases from RTOG 9202 5
. Abnormal p53 was noted in 22% of these cases, and was associated with cause-specific survival (p=0.014) and risk of distant metastasis (p=0.013). In the subgroup of patients treated with short term ADT, there was a correlation between p53 status and cause-specific survival (p=0.004). When patients were divided into subgroups according to p53 status, only the subgroup of patients with abnormal p53 showed significant association between the assigned treatment and cause-specific survival (p<0.01). Unfortunately, because all patients on RTOG 9202 received ADT, the question of a possible unfavorable interaction between abnormal p53 expression and the use of short term ADT compared to EBRT alone (raised in the previous analysis based on RTOG 8610) could not be resolved.
DNA-Ploidy was evaluated using a cohort of 149 men (33% of the total number) treated on RTOG 8610 3
. From this cohort 50% were treated with EBRT alone, and 50% with EBRT and short-term ADT. DNA ploidy was independent of other pretreatment variables. The 5-year overall survival was 70% in those with diploid tumors and 42% for non-diploid tumors. Although, the possession of a non-diploid tumor was associated with a reduced overall survival, there was no correlation with distant metastasis. The investigators postulated that patients with non-diploid tumors might be less responsive to salvage ADT and that the use of short term ADT might not be advisable in patients with non-diploid tumors.
Based on a subset of patients treated on RTOG 8610, loss of p16 expression, was associated with an increased risk of local failure, distant metastasis and disease-specific survival (p<0.01; p<0.03; and p<0.01, respectively) and there was a borderline association with on overall survival of (p=0.07) 6
. In an analysis of 612 patients from RTOG 9202, reduced expression was associated with an increased rate of distant metastases (p<0.04) 7
. Among patients with high immunostaining for p16, the use of long term ADT was associated with an increase in cause specific survival and a decreased incidence of distant metastasis compared to short-term ADT (p<0.001, p<0.01). This may suggest that p16 expression might be associated with increased hormonal sensitivity.
Ki-67 was the next marker evaluated by the RTOG using tissue from RTOG 8610. Diagnostic material from 108 patients was available for Ki-67 analysis with 60 patients treated with EBRT alone, and 48 also receiving short-term ADT 8
. Ki-67 staining index </=3.5% and >3.5%, was associated with a 5-year risk of distant metastasis of 13.5% and 50.8% (p=0.0005), a 5-year risk of disease specific survival of 97.3% and 67.7% (p=0.0039), and a five year overall survival rate of 70 and 55% (p=0.17). These trends were confirmed in multivariate analyses. An additional assessment of Ki-67 was also made using tissue from 537 patients treated on RTOG 9202 9
. When analyzed as a continuous variable, Ki67 staining index was associated with the risk of distant metastasis (P <0.0001), disease specific survival (p<0.0001), and overall survival (p< 0.01), and was the most significant predictor of the first two endpoints. Based on a subset analysis, the authors hypothesized that there may be a subgroup of patients who do not require long term ADT. This observation suggests that Ki-67 might be useful in selecting patients for short term ADT and stratifying patients placed on future trials.
The RTOG also evaluated the association between MDM2 expression (an oncoprotein that promotes p53 degradation) and outcomes using tissue from RTOG 8610 10
. Adequate archival diagnostic tissue specimens from 108 patients with MDM2 overexpression classified as having > 5% nuclear staining was seen in 44% (n = 47) of specimens. By multivariate analysis there was trend for a relationship with the risk of distant metastasis at 5 years (P = 0.06). A more comprehensive and promising study combining MDM2 with Ki-67 based on patients treated on RTOG 9202 is near completion (but not yet available). The results indicate that MDM2 compliments Ki-67, is much stronger a predictor of outcome than p53 and the combination of MDM2 and Ki-67 has promise in identifying men at a particularly high risk of distant metastases.
Bcl-2 and Bax expression were also evaluated using tissue form RTOG 8610 11
. Suitable diagnostic tissue was available from 119 (26%) patients for bcl-2 and 104 (23%) for bax analysis. Bcl-2 over expression was exhibited in 26% (n = 30) and abnormal bax expression in 47% (n = 49) of cases. Based on this analysis, neither were found to be related to outcomes. A follow-up investigation was carried out using tissue from 502 patients for Bcl-2 and 343 patients for Bax treated on RTOG 9202 12
. Bcl-2 was positive in 45.6% cases, and Bax expression altered in 53.9% cases. The combination of negative Bcl-2/normal Bax expression was related to reduced biochemical failure (p= 0.036), particularly among those who received short term ADT suggesting that long term ADT might be advised when either Bcl-2 or Bax is abnormally expressed.
A quantitative assessment of CAG base pair repeats on the androgen receptor (AR) gene was also performed on a subset of patients treated on RTOG 8610 13
. CAG repeats were measured in 94 tumor specimens and did not significantly influence either local control, the risk of metastasis, cause specific or overall survival. Patients with short repeats who received short term ADT did seem to have a higher local control rate, however.
Cox-2 expression was also evaluated using tissue from 586 men treated on RTOG 9202 who had sufficient tissue for immunohistochemical staining and image analysis 14
. The intensity of Cox-2 staining was predictive of the risk of distant metastasis (p=0.0004) and the risk of biochemical failure using both the ASTRO and the Phoenix definitions (p=0.008 and p=0.014), particularly among those who were treated with short term ADT.
Stat-3 expression was evaluated in a subset of 62 patients who had sufficient tissue from RTOG 8610 15
. Activated STAT3 was inversely correlated with the development of distant metastasis (p=0.04), but not survival or local control although due to the small sample size this conclusion has to be cautiously interpreted.
Polymorphisms in the androgen receptor CYP3A4 were evaluated for a subset of patients treated on RTOG 9202 to understand how variation polymorphisms in CYP3A4 correlated with outcomes and race 16
. Tissue from 56 men African-American origin and 54 of European-American patients was studied. There was a strong association between race and CYP3A4 polymorphisms with 75% of European-Americans having the Wild Type compared to only 25% of African-American men (p<0.0001) but there was no association between CYP3A4 polymorphisms studied and outcomes.
Archival diagnostic tissue samples from 80 patients treated on RTOG 8610 was used to study the predictive value of protein kinase A RI-alpha (PKA) expression 17
. On multivariate analyses, there was a correlation between overexpression and increased biochemical failure (p=0.03), increased distant metastasis (p=0.018), and a trend for increased cause specific mortality (p=0.08). The analysis PKA expression to RTOG 9202 patient outcome is nearing completion and validates the findings from RTOG 8610.
Several conclusions can be reached based on these hypothesis-generating studies. Despite these interesting observations taken as a whole the major conclusion remain. Biomarkers as a major predictor of outcome are not yet ready for routine use in clinical practice. However, the relationships observed are promising. A model is being developed that incorporates the key markers identified in cases from RTOG 92-02; preliminary evidence indicates that such a model will add significantly to classic clinical, pathologic and treatment-related covariates in predicting distant metastasis at 10 years. The preliminary findings of these studies support the need for validation studies (which are in progress). As is shown in ., the most robust observations relate to the clinical relevance of p16, Ki-67, MDM2, Cox-2 and PKA, with most of these appearing to be useful in identifying subsets of patients who do not appear to benefit from LTADT.