While absent or weakly expressed in benign prostatic glands (Figure ), RBM3 was clearly up-regulated in prostatic intraepithelial neoplasia (PIN) (Figure ) and in invasive carcinoma RBM3 was expressed in various fractions and intensities (Figure ) with 33 (37.5%) cases lacking RBM3 expression (Figure ). A progression sequence with up-regulated RBM3 expression in PIN and retained high RBM3 expression in the invasive component is illustrated in Figure . There was no obvious heterogeneity in the staining pattern between duplicate tissue cores.
Figure 1 RBM3 expression in normal prostatic epithelium, prostatic intraepithelial neoplasia and in prostate cancer. Immunohistochemical images of RBM3 expression in (A) normal prostatic epithelium, (B) prostatic intraepithelial neoplasia and invasive prostate (more ...)
RBM3 staining distribution in prostate cancer. Distribution of nuclear RBM3 staining in invasive prostate cancer, denoted as nuclear score (fraction × intensity).
Example of RBM3 up-regulation in prostate cancer progression. Immunohistochemical image depicting up-regulated RBM3 expression in (A) PIN and (B) adjacent invasive component with high RBM3 expression.
CRT analysis suggested an optimal cutoff point at NS > 2 to determine the impact of RBM3 expression on BCR free survival and PFS. While there was no association between RBM3 expression and conventional clinicopathological parameters (Additional File 2
), Kaplan Meier analysis demonstrated that high expression of RBM3 was associated with a significantly prolonged time to BCR (p
= 0.004) and clinical progression (p
= 0.004) (Figure ). These associations were confirmed in Cox univariate analysis (HR 0.56, 95% CI: 0.34-0.93, p
= 0.024 for BCR and HR 0.09, 95% CI: 0.01-0.71, p =
0.021 for clinical progression) and remained significant in multivariate analysis, adjusted for preoperative PSA level, Gleason score and presence or absence of extracapsular extension, seminal vesicle invasion and positive surgical margins (HR 0.41, 95% CI: 0.19-0.89, p
= 0.024 for BCR and HR 0.06, 95% CI: 0.01-0.50, p =
0.009 for clinical progression) (Table ). These significant associations were retained in both univariate and multivariate analysis also when the few patients who received adjuvant radiotherapy or hormonal therapy were excluded from the analysis (data not shown). Cytoplasmic RBM3 expression was not prognostic (data not shown).
Prognostic value of RBM3 expression in prostate cancer. Kaplan Meier curves visualizing the impact of RBM3 expression on (A) biochemical recurrence free and (B) progression free survival.
Cox univariate and multivariate analysis of biochemical recurrence free survival and progression free survival according to RBM3 expression
Interpretation and conclusions
Our results demonstrate that RBM3, while rarely expressed in normal prostatic gland epithelium, is up-regulated in PIN and invasive prostate cancer, and that patients with tumours expressing high nuclear levels of RMB3 have a significantly prolonged time to biochemical recurrence and clinical progression, also after adjustment for conventional prognostic factors. These findings are in line with previous findings in breast cancer [11
], ovarian cancer [15
], malignant melanoma [16
] and colorectal cancer (Hjelm et al, Proteomics Clinical Applications, in press). Thus, there are increasing evidence for RBM3 being a biomarker of good prognosis in multiple cancer forms. The mechanisms behind these findings remain to be elucidated, an undertaking that might be somewhat challenging as current in vitro data point towards a proto-oncogenic role for RBM3 [8
] and, hence, do not readily seem to support the clinical situation. However, the common hypothesis that oncogene activation is associated with an aggressive tumour phenotype does not always hold true, as well exemplified by the association between microsatellite instability and good prognosis in colorectal cancer [20
]. It is evident that RBM3, while sparsely expressed in normal tissue, is up-regulated in most cancer forms and their pre-invasive stages in vivo
. In light of the recently proposed association between RBM3 and DNA integrity in ovarian cancer [17
], it could be speculated that RBM3 might play an important role in promoting early stages of tumourigenesis by interfering with the anti-cancer barrier provided by various DNA damage checkpoint mechanisms [21
]. On the other hand, once a tumour has been established, an attenuated capability of DNA-damage response caused by RBM3 over expression might hinder the pressure for selection of more malignant clones [21
]. Further studies are warranted to explore these associations.
In this study, we used a well-validated monoclonal antibody for detection of RBM3 expression [15
]. Immunohistochemistry has several advantages compared to other assays in that it can easily be incorporated into clinical protocols and allows for assessment of the subcellular localization of proteins, which might have important prognostic implications. Our results indicate that immunohistochemical assessment of RBM3 expression in formalin-fixed paraffin embedded tumour samples could be a useful tool for prognostication and treatment stratification of prostate cancer patients. Since this was a small study these findings should be further validated in larger studies, preferably tumours from prospective, clinical trials.