This is the first comprehensive immunohistochemical analysis of the expression of several class I HDAC proteins (1, 2 and 3) in urothelial carcinoma. In our study, we found all three isoforms in a relevant amount of all investigated urothelial tumours. HDAC-1 and HDAC-2 were highly associated with high-grade superficial papillary bladder tumours. Additionally, high expression levels of HDAC-1 showed a tendency towards a shorter PFS.
So far, little was known about class I HDAC expression pattern in urothelial cancer . According to the Proteinatlas (http://www.proteinatlas.org), HDAC-1 to -3 expression levels are moderate at most in urothelial cancer . In previous expression arrays HDAC-2 and -3 showed higher expression levels in urothelial cancer than in normal urothelial tissue [2, 9, 11]. Expression array data from another study by Wild et al. demonstrated an upregulation of HDAC-1 in bladder cancer compared to normal urothelial tissue . On the contrary, published data from other groups did not reveal any difference of class I HDAC expression between urothelial cancer and normal urothelium in microarray data [24, 25]. In accordance with these findings a study from Xu reported no difference in immunohistochemical expression of HDAC-2 in human bladder cancer tissue (142 cases) compared to normal urothelial tissue (23 cases) .
In a recent study, Niegisch and colleagues were able to show upregulation of HDAC-2 mRNAs in a subset of tested tumours compared to normal urothelium [9, 11, 17]. However, only 24 tumour tissues and 12 normal samples were tested.
Our study is the first attempt to test the immunohistochemical expression of class I HDACs in a large cohort of patients with bladder cancer. As class I HDACs can be detected in a relevant group of urothelial cancer, they may therefore be relevant in pathophysiology and as target proteins for treatment.
Besides the distinct presence of class I HDACs in urothelial cancer, high expression levels of HDAC-1 and -2 were associated with stage and grade of this tumours. Overexpression of HDACs has been found in several other solid tumours such as prostate and colon cancer [7, 26, 27]. High expression levels of class I HDACs correlated with tumour dedifferentiation and higher proliferative fractions (measured by Ki-67) in urothelial carcinoma, which is in line with in vitro studies showing that high HDAC activity leads to tumour dedifferentiation and enhanced tumour cell proliferation [28–30]. Despite the growth inhibitory effects of HDAC-i demonstrated in various cell lines including bladder cancer cells, a broad expression analysis of this attractive target has not been conducted yet [12–16, 31, 32].
To the best of our knowledge, this is the first study analysing HDAC-1, -2 and -3 expression in bladder cancer and its association to prognosis. In our study HDAC-1 was found to be of rough prognostic relevance in pTa and pT1 tumours. High expression levels of class I HDACs have been found to be of prognostic relevance in other tumour entities before. Other study groups previously reported the association of class I HDACs with more aggressive tumours and even shortened patient survival in prostate and gastric cancer [7, 33]. Our findings suggest that HDAC-1 may have a role in prognosis of superficial urothelial tumours.
In our work the rate of Ki-67 positive tumour cells was highly associated with tumour grade, stage, and a shorter PFS. A substantial amount of research has demonstrated the prognostic role of Ki-67 in urothelial cancer; its prognostic value and its association with pathological parameters and prognosis could be shown in several studies [34–36]. These findings are in line with our work and confirm the representativeness and validity of this TMA-construct. Furthermore, we observed a strong correlation between the proliferation index (Ki-67) and all three investigated HDACs. The connection between HDAC expression and Ki-67 observed in urothelial carcinoma has already been demonstrated for prostate, renal and colorectal cancer in previous studies [7, 19, 37].
Additionally, intravesical instillation of HDAC-i may have a potential as chemopreventive agent to treat superficial bladder cancer, as up to 50% of superficial tumours showed high expression levels of HDACs. However, it is not clear whether HDAC protein expression as assessed by immunohistochemistry is a predictor for treatment response to HDAC-i. Thus, additional studies are needed to clarify the role HDAC-i in non-invasive urothelial cancer.
Our study has several limitations, including its retrospective design and the use of immunohistochemical methodology, which has inherent limitations, including scoring of staining. We used a standardized and well-established semiquantitative scoring method in accordance with previous publications to reduce variability. In addition, the proportion of muscle-invasive bladder cancer was limited and as a consequence we cannot draw any conclusion for this subgroup of tumours. Therefore future research should also try to assess whether class I HDACs have a prognostic value in locally advanced invasive or metastatic urothelial cancer.