Histone deacetylase (HDAC)-1, −2, −4 and −6 expression in human pancreatic adenocarcinoma: associations with clinicopathological parameters, tumor proliferative capacity and patients’ survival

Seventy pancreatic ductal adenocarcinoma specimens obtained from equal number of patients who underwent surgical resection due to pancreatic cancer were included in this study. The study was approved by the institutional ethical committee of the Medical School of the University of Athens. Informed consent to use their biological samples and clinical data for research purposes was signed by all patients under study. None of the patients received any kind of anti-cancer treatment prior to surgery. Forty-four of the patients were men (62.9 %) and 26 women (37.1 %), with a mean age of 66.77 ± 8.94 years (range 33–84 years). The cases were classified based on the World Health Organization criteria for histological grading as: well in 9 (12.9 %); moderately in 51 (72.8 %); poorly differentiated in 10 (14.3 %) [32]. Tumor staging was assessed using the 5th edition of the Tumour, Node, Metastasis (TNM) and the American Joint Committee on Cancer (AJCC) Grouping system [33, 34]. In fact, tumors were classified as: T1 in 4 (5.7 %), T2 in 8 (11.4 %), T3 in 48 (68.6 %) and T4 in 10 (14.3 %) cases. Thirty-three (47.1 %) were lymph node negative (N0), and 37 (52.9 %) were regional lymph node positive (N1). Organ metastasis was noted in 4 (5.7 %) out of 70 patients examined. According to the AJCC classification, 10 (14.3 %) cases were characterized as stage I, 46 (65.7 %) as stage II, 10 (14.3 %) as stage III and 4 (5.7 %) as stage IV. The patients were followed up until death for a time interval of 4 up to 21 months with a mean survival time of 8.69 ± 3.57 months. Overall survival was defined as the time interval between the date of surgery and the date of death due to pancreatic adenocarcinoma. At the time of the last follow-up, all patients had died from the disease.

Immunostainings for HDAC-1, −2, −4 and −6 were performed on individual formalin-fixed, paraffin-embedded pancreatic adenocarcinoma tissue sections using rabbit polyclonal anti-HDAC-1 (H-51, sc-7872, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-HDAC-2 (H-54, sc-7899, Santa Cruz Biotechnology) IgG antibodies and mouse monoclonal anti-HDAC-4 (A-4, sc-46672, Santa Cruz Biotechnology) IgG_2b and anti-HDAC-6 (D-11, sc-28386, Santa Cruz Biotechnology) IgG_2a antibodies. Briefly, 4 μm thick tissue sections were dewaxed in xylene and were brought to water through graded alcohols. Antigen retrieval was performed by microwaving slides in 10 mM citrate buffer (pH 6.1) for 15 min (min) at high power, according to the manufacturer’s instructions. To remove the endogenous peroxidase activity, sections were then treated with freshly prepared 0.3 % hydrogen peroxide in methanol in the dark, for 30 min, at room temperature. Non-specific antibody binding was blocked using Sniper, a specific blocking reagent for mouse and rabbit primary antibodies (Sniper, Biocare Medical, Concord, California, USA) for 5 min. The sections were incubated for 1 h (h), at room temperature, with the primary antibodies against HDAC-1, −2, −4 and −6 diluted 1:200 in phosphate buffered saline (PBS) according to the manufacturer’s instructions. Sections were then incubated at room temperature with biotinylated linking reagent (Biocare Medical) for 10 min, followed by incubation with peroxidase-conjugated streptavidin label (Biocare Medical) for 10 min. The resultant immune peroxidase activity was developed using a DAB substrate kit (Vector Laboratories, California, USA) for 10 min. Sections were counterstained with Harris’ hematoxylin and mounted in Entellan (Merck, Darmstadt, Germany). Appropriate negative controls were performed by omitting the primary antibody and/or substituting it with an irrelevant anti-serum. As positive control, mobile tongue squamous and thyroid carcinoma tissue sections with known increased HDAC-1, −2, −4 and −6 expression were used [17, 18]. Isotype controls were not performed. The tumour cells’ proliferative capacity was assessed by Ki-67 immunohistochemical expression, as previously described [17, 18].

Immunohistochemical evaluation was performed by counting at least 1000 tumor cells in each case by two independent observers (S.T. and E.P.) blinded to the clinical data, with complete observer agreement. Specimens were considered “positive” for HDAC-1, −2, −4 and −6 when more than 5 % of tumor cells within the section were positively stained. HDAC-1, −2, −4 and −6 immunoreactivity was scored according to the percentage of positive tumor cells as 0: negative staining- 0–4 % of cells positive; 1: 5–24 % of cells positive; 2: 25–49 % of cells positive; 3: 50–100 % of cells positive, and its intensity as 0: negative staining, 1: mild staining; 2: intermediate staining; 3: intense staining. Finally, the expression of HDAC-1, −2, −4 and −6 was classified as low; if the total score was 0 or 2 and high; if the total score was ≥3 [17, 18]. In this way, we ensure that each group has a sufficient and more homogeneous number of cases in order to be comparable with the other groups [17, 18]. Ki-67 immunoreactivity was classified according to the percentage of positively stained tumor nuclei exceeding the mean percentage value into two categories (below and over median value), as previously reported [17, 18].

Chi-square test was used to assess the associations of HDAC-1, −2, −4 and −6 proteins’ expression with clinicopathological variables. Survival curves were constructed using the Kaplan-Meier method and the differences between the curves were compared by the log rank test. A Cox proportional-hazard regression model was developed to evaluate the association between the potential prognostic marker and overall survival, at multivariate level. A p-value less than 0.05 was considered the limit of statistical significance. SPSS for Windows Software was used for all analyses (SPSS Inc., 2003, Chicago, USA).

Forty-five (64.3 %) out of 70 pancreatic adenocarcinoma cases were found HDAC-1 positive. The pattern of HDAC-1 distribution was nuclear in all positive cases (Fig. 1a). High HDAC-1 expression was noted in 28 (40.0 %) out of 70 pancreatic adenocarcinoma cases. Non-neoplastic sites of pancreatic tissues were found negative for HDAC-1 (data not shown).

In crosstabulation, high HDAC-1 expression was significantly associated with increased tumor proliferative capacity (Table 1, p = 0.0238). High HDAC-1 expression was borderline more frequently observed in pancreatic adenocarcinoma patients with absence of lymph node metastases (Table 1, p = 0.0632). High HDAC-1 expression was also more frequently observed in pancreatic adenocarcinoma patients presenting smaller tumor size and earlier histopathological stage, at a non significant level though (Table 1, p = 0.1544 and p = 0.1127, respectively).

Kaplan-Meier survival curves indicated that pancreatic adenocarcinoma patients presenting high HDAC-1 expression had significantly longer survival times compared to those with low expression (Fig. 2a, Table 2, log-rank test, p = 0.0022). In multivariate analysis, histopathological stage but not HDAC-1 expression was identified as independent prognostic factor of patients’ survival (Table 3, Cox-regression analysis, p < 0.001and p = 0.0521, respectively).

Forty-nine (74.2 %) out of 70 pancreatic adenocarcinoma cases were found HDAC-2 positive. The pattern of HDAC-2 distribution was nuclear in all positive cases (Fig. 1b). High HDAC-2 expression was noted in 35 (50.0 %) out of 70 pancreatic adenocarcinoma cases. Non-neoplastic sites of pancreatic tissues were found negative for HDAC-2 (data not shown).

In crosstabulation, HDAC-2 expression was not significantly associated with any clinicopathological parameters examined (Table 1). High HDAC-2 expression was more frequently observed in pancreatic adenocarcinoma patients presenting smaller tumor size, absence of lymph node metastases and earlier histopathological stage, at a non significant level though (Table 1, p = 0.2046, p = 0.2312 and p = 0.2320, respectively). Kaplan-Meier survival curves indicated that pancreatic adenocarcinoma patients presenting high HDAC-2 expression had marginally longer survival times compared to those with low expression (Fig. 2b, Table 2, log-rank test, p = 0.0634).

Concomitant HDAC-1/HDAC-2 expression was not significantly associated with any clinicopathological parameters (p > 0.05, data not shown). Kaplan-Meier survival curves indicated that pancreatic adenocarcinoma patients presenting concomitant high HDAC-1/HDAC-2 expression had significantly longer survival times compared to those with low expression (log-rank test, p = 0.0255). Concomitant HDAC-1/HDAC-2 expression was not remained significant in multivariate analysis (p > 0.05, data not shown).

Forty-seven (69.1 %) out of 70 pancreatic adenocarcinoma cases were found HDAC-2 positive. The pattern of HDAC-4 distribution was cytoplasmic in all positive cases (Fig. 1c). High HDAC-4 expression was noted in 34 (48.6 %) out of 70 pancreatic adenocarcinoma cases. Non-neoplastic sites of pancreatic tissues were found negative for HDAC-4 (data not shown).

In crosstabulation, high HDAC-4 expression was significantly associated with the absence of organ metastases (Table 4, p = 0.0453). High HDAC-4 expression was more frequently observed in pancreatic adenocarcinoma patients with absence of lymph node metastases and increased tumor proliferative capacity, at a non significant level though (Table 4, p = 0.0571 and p = 0.0576, respectively). High HDAC-4 expression was also more frequently observed in pancreatic adenocarcinoma patients presenting earlier histopathological stage, at a non significant level though (Table 4, p = 0.2818). HDAC-4 expression was not associated with patients’ survival (Fig. 2c, Table 2, log-rank test, p = 0.5061).

Forty-one (63.1 %) out of 70 pancreatic adenocarcinoma cases were found HDAC-6 positive. The pattern of HDAC-6 distribution was cytoplasmic in all positive cases (Fig. 1d). High HDAC-6 expression was noted in 31 (44.3 %) out of 70 pancreatic adenocarcinoma cases. Non-neoplastic sites of pancreatic tissues were found negative for HDAC-6 (data not shown).

In crosstabulation, HDAC-6 expression was significantly associated with earlier histopathological stage (Table 2, p = 0.0115). High HDAC-6 expression was more frequently observed in pancreatic adenocarcinoma patients with smaller tumor size and absence of organ metastases, at a non significant level though (Table 4, p = 0.0864 and p = 0.0663, respectively). High HDAC-6 expression was more frequently observed in younger and female pancreatic adenocarcinoma patients, as well as in those with absence of lymph node metastases, at a non significant level though (Table 4, p = 0.2920, p = 0.2157 and p = 0.1026, respectively).

Kaplan-Meier survival curves indicated that pancreatic adenocarcinoma patients presenting high HDAC-6 expression had significantly longer survival times compared to those with low expression (Fig. 2d, Table 2, log-rank test, p = 0.0113). In multivariate analysis, histopathological stage but not HDAC-6 expression was identified as independent prognostic factor of patients’ survival (Table 5, Cox-regression analysis, p < 0.001and p = 0.2193, respectively).