Based on the fact that RelA p65 is Bioactive compound a putative target of HDIs, we addi tionally tested the effects of the two well known HDIs SAHA and VPA on RelA p65 activity in vitro. Methods Study Population Tissue samples from 81 patients who underwent partial pancreaticoduodenectomy for primary pancreatic ductal adenocarcinoma at the Charit�� University Hospital between 1991 and 2000 were used in this study. The study has been approved by the Charit�� University Ethics Com mittee under the title Retrospektive Untersuchung von Gewebeproben mittels immunhistochemischer FArbung und molekularbiologischer Methoden at the 20th of Sep tember 2004. Median age of patients with pancreatic cancer was 66 years. Follow up data regarding over all survival were available for all patients.
Within the fol low up time, 64 patients died after a median follow up time of 11. 7 months. Median follow up time of patients still alive at the endpoint of analysis was 44. 0 months. Cases were staged according to TNM Classifica tion of Malignant Tumours. 6th edition and cases were graded as recommended by the WHO. Distribution of clinico pathological factors in the study cohort is given in Additional file 1. Immunohistochemical staining and histopathological examination RelA p65 expression patterns had been determined in 78 of the 81 cases in a previous study. For immunohis tochemical detection of HDAC isoforms on tissue sam ples, prediluted polyclonal rabbit IgG antibody directed against HDAC1, mono clonal mouse IgG antibody directed against HDAC2 and monoclonal mouse IgG antibody directed against HDAC3 were used on 5 m paraffin sections.
Antibody specificity had already been ascertained in a previous study. Immu nohistochemistry was done as previously described. Nuclear staining of HDAC isoforms was scored by apply ing a semiquantitative immunoreactivity scoring system, as previously described. Briefly, intensity of staining as well as percentage of cells stained was evalu ated separately. Cilengitide The IRS for each individual case ranging from 0 to 12 was calculated by multiplication of the inten sity and frequency scores. Cases exhibiting an IRS from 0 6 were combined in one group, cases with an IRS of more than 6 were combined in a HDAC positive group. In addition, the patients were grouped according to their overall class I HDAC expression pattern. Staining of tis sue slides was evaluated by an experienced pathologist who was blinded towards patient characteristics and outcome. Statistical evaluation Statistical analyses were carried out with SPSS 15. 0 and GraphPad Prism 4. 0. The significance of correlations between HDAC isoform staining patterns and clinico pathological data was assessed by Fishers exact test and ?2 test for trends.