High nuclear expression levels of histone-modifying enzymes LSD1, HDAC2 and SIRT1 in tumor cells correlate with decreased survival and increased relapse in breast cancer patients

The patient population was a retrospective cohort of female breast cancer patients (TNM: I-III) who underwent primary tumor resection at the Leiden University Medical Center (LUMC) between 1985 and 1996 (n = 822), as described previously [14]. Patients with bilateral tumors or a prior history of cancer (other than basal cell carcinoma or cervical carcinoma in situ) were excluded from the study. The following data were retrieved and used as covariates in multivariate analyses: age, tumor size, nodal status, expression of estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor 2 (HER2), tumor grade, histological type, local and systemic therapy, survival time, and time until tumor relapse. All tumors were graded and histologically classified according to pathological standards by an experienced breast cancer pathologist (V.S.). The study was conducted with anonymized patient data according to Dutch law and in agreement with the Dutch Code of Conduct: “Proper Secondary Use of Human Tissue in the Netherlands” (Federation of Medical Scientific Societies, the Netherlands, http://​www.​federa.​org/​sites/​default/​files/​bijlagen/​coreon/​codepropersecond​aryuseofhumantis​sue1_​0.​pdf). The specific section is paragraph one of chapter eight on page 43 and therefore we did not ask for approval of an ethics committee [15], and according to the REMARK guidelines [16].

Formalin-fixed paraffin-embedded (FFPE) tumor tissue of 701 patients, of whom tumor tissue was available, was included into a tissue microarray (TMA), as described previously [14]. For each patient, three cores of tumor tissue were included. For 261 breast cancer patients, of whom normal epithelial breast tissue was available, three cores of normal breast tissue were included in separate TMA blocks.

TMA sections were cut (4 μm) and processed for immunohistochemistry (IHC). The antibodies that were used for IHC were validated by several other research groups: anti-LSD1 (ab17721, mouse, Abcam, Cambridge, United Kingdom) [17, 18], anti-HDAC2 (ab39669, rabbit, Abcam) and anti-SIRT1 (ab32441, rabbit, Abcam) [19]. The IHC was performed using a standard protocol [20]. Briefly, tissues were deparaffinized in xylene and rehydrated in a series of graded alcohol. Antigen retrieval was performed by heating the sections for 10 min in sodium-citrate buffer at 95°C (pH 6.0). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide solution for 20 minutes. Incubation, with an optimized concentration of the antibodies described, was performed overnight at room temperature. Envision + peroxidase labelled polymer rabbit or mouse (Dako, Glostrup, Denmark) and DAB + liquid substrate chromogen system (Dako) were used for visualization of the expression levels. Counterstaining was performed using haematoxylin and dehydration was performed using graded alcohol and xylene.

The scoring of the immunohistochemical staining was performed by two investigators (A.S. and G.D.), who were blinded for the clinicopathological data. The percentage of positive stained tumor cell nuclei was scored in each of the tissue cores, from 0-100% with 10% increments. The second observer scored 30% of the tissue cores in order to determine consistency in quantification, which was tested with Cohen’s kappa coefficient for inter-observer variability. A Cohen’s kappa coefficient >0.6 was considered as substantial agreement. In addition to tumor tissues, stained normal epithelial breast tissue cores were also evaluated using the same scoring criteria as described above.

Data were analyzed using SPSS 20.0 for Windows (SPSS Inc., Chicago, Illinois, United States of America). The paired student’s t-test was used to compare expression levels in tumor breast tissues and their corresponding normal epithelial tissues of 60 individual patients. The one-way ANOVA method was used for calculation of differences in expression levels between the TNM tumor stages (I-III) for LSD1, HDAC2 and SIRT1. For survival analyses, the patients were divided into a low and high expression category based on the median percentage positive tumor cell nuclei per enzyme. The Cox proportional hazards model was used for univariate and multivariate survival analyses. Kaplan-Meier (KM) curves and cumulative incidence curves were plotted to graphically show differences in patient survival and tumor relapse between the groups with different expression levels, respectively. For the uni- and multivariate analyses, only patients with nuclear staining data for all three enzymes and all covariates available, complete case analysis, were used in the statistical analyses (n = 460). Data were censored when patients were alive or free of relapse at their last follow-up date (lastly march 2013). Overall survival (OS) was defined as the time from date of surgery until death from any cause. Relapse-free survival (RFS) was defined as the time from surgery until the occurrence of a local, regional or distant tumor relapse or death by cancer. The Pearson Chi-square method was used to test for correlations between the combined expression levels of LSD1, HDAC2 and SIRT1 and clinical parameters. The low expression group was used as a reference in the single marker analyses. Low expression of all three markers was used as reference in the analyses of the combined expression levels. For the analyses of the combined expression levels of the markers, the patients were divided into four categories as follows: all enzymes below-median expression (‘all-low’), one enzyme above-median expression, two enzymes above-median expression and all three enzymes above-median expression (‘all-high’). We performed a Chi-square test between the four patients groups and all variables used as covariates, which are well-known independent prognostic factors in breast cancer and we corrected for those covariates in the multivariate analyses. For all analyses, a two-sided p-value ≤0.05 was considered statistically significant.

Table 1 shows the clinicopathological data of the breast cancer patients (n = 460) used for the statistical analyses of the three markers. The mean follow-up time was 11.8 years (range: 0.16-27.55 years) and the mean age at diagnosis was 58.3 years (range: 23–89 years). Percentages of positive nuclei for LSD1, HDAC2 and SIRT1 in the tumor and normal tissue cores were determined by IHC. Figure 1 shows representative pictures of normal breast tissue cores immunohistochemically stained individually for each enzyme, as well as representative pictures of breast cancer tissue cores with expression above and below median for each of the enzymes. The brown color is the amount of expression of the enzyme. The median percentages of positive tumor nuclei, used for the statistical analyses, were 85% for LSD1, 80% for HDAC2 and 70% for SIRT1. Cohen’s kappa coefficient was calculated to determine the inter-observer variability. The kappa coefficients for scoring of the tumor tissues were 0.664 for LSD1 and 0.627 for SIRT1. Both kappa coefficients were considered as substantial agreement between the observers. For staining of HDAC2 in tumor tissues, the kappa for scoring of the tumor tissue was not considered as substantial agreement. Therefore, a re-evaluation of the scoring was performed by the two observers until agreement was reached. For normal tissues the kappa coefficients were 0.693 for LSD1, 0.628 for HDAC2 and 0.605 for SIRT1, which were all considered as substantial agreement as well. The mean percentage of positive nuclei in the cores determined for each patient by the first observer, was used for survival analyses. Figure 2 shows the expression levels of LSD1, HDAC2 and SIRT1 in normal breast tissues compared to tumor tissues. Analyses of paired tumor and normal tissues showed an increased expression of LSD1 and SIRT1 in tumor tissues compared to normal tissues (both p < 0.001). HDAC2 expression did not significantly differ in tumor tissues compared to normal tissues (p = 0.4).

To investigate whether expression of each of the histone-modifying enzymes was related to the TNM tumor stage, the mean percentage of positive tumor nuclei was plotted against tumor stage. Figure 3 shows the percentage of positive nuclei in each tumor stage (I-III) for LSD1, HDAC2 and SIRT1. A one-way ANOVA analysis showed significant differences between the tumor stages for LSD1 (p < 0.001) and SIRT1 (p = 0.04) (Figures 3A and 3C). With higher expression in patients diagnosed with a higher tumor stage. HDAC2 did not show a significant difference between the tumor stages (p = 0.4) (Figure 3B).

Univariate analyses showed significant differences in patient survival and tumor relapse between patients with high and low nuclear expression of LSD1 (OS: p = 0.002, HR = 1.42, 95% CI = 1.13-1.77; RFS: p = 0.001, HR = 1.55, 95% CI = 1.20-1.99) and SIRT1 (RFS: p = 0.03, HR = 1.32, 95% CI = 1.03-1.70) (Figures 4A, 4C and 4D). No significant differences were observed for HDAC2 expression (OS: p = 0.1, HR = 1.23, 95% CI = 0.99-1.54; RFS: p = 0.1, HR = 1.25, 95% CI = 0.98-1.61) (Figure 4B and 4D). Multivariate analyses of the expression levels for individual markers showed a significant difference in RFS for SIRT1 (p = 0.02, HR = 1.34, 95% CI = 1.04-1.74) with shorter RFS in the high expression group (Figure 4D). No significant differences were observed for HDAC2 (OS: p = 0.6, HR = 1.07, 95% CI = 0.85-1.34; RFS: p = 0.2, HR = 1.16, 95% CI = 0.90-1.50) and LSD1 (OS: p = 0.2, HR = 1.18, 95% CI = 0.94-1.50; RFS: p = 0.1, HR = 1.23, 95%CI = 0.94-1.60) in the multivariate analyses (Figure 4D).

Since the three enzymes work together in one complex, we hypothesized that the combined expression levels of the three histone-modifying enzymes is a stronger predictor for patient survival and tumor relapse than expression of individual enzymes. Survival analyses of OS and RFS showed that the combined expression level of LSD1, HDAC2 and SIRT1 in breast tumors was more predictive for patient survival and tumor relapse than each of the individual markers separately in both univariate and multivariate analyses (Figure 5). Chi-square analyses showed that there were significant differences between the four patient groups in ER (p = 0.019), PgR (p = 0.007), tumor grade (p < 0.001) and systemic therapy (p = 0.010), for which we corrected in the multivariate analyses. Multivariate analyses of the combined marker expression levels showed that patients with high expression level of all three markers had a shorter OS compared to patients with low expression of all the enzymes (p = 0.03, HR = 1.49, 95% CI = 1.07-2.08) (Figure 5C). For RFS the HR was 1.68 (p = 0.006, 95% CI = 1.16-2.44) in the ‘all-high’ expression group versus the ‘all-low’ expression group (Figure 5C). This result indicated that patients with high expression of all three enzymes have a shorter RFS compared to patients with one or more enzymes with a low expression level.

We tested if there was a correlation between the combined expression levels of the three enzymes and tumor differentiation, a marker of aggressive tumors, in the whole study population. Indeed, a significant correlation between these expression levels and tumor differentiation was found (p < 0.001; Table 2). The results showed that 24% of the patients with low expression of all three enzymes had a well-differentiated tumor and only 12% of the patients with high expression of all three enzymes had a well-differentiated tumor. A low differentiation grade was found in 21% of patients with low expression of LSD1, HDAC2 and SIRT1 and 43% of the patients with high expression of all three enzymes had a low grade of tumor differentiation. In addition, we investigated the relation between the combined expression levels of LSD1, HDAC2 and SIRT1 and tumor cell proliferation, assessed by ki-67 expression, which is another marker of aggressive tumors. Ki-67 expression levels were determined by IHC previously in our study cohort [21] and data were available for 423 of 460 patients (92%). A significant correlation was found between the expression of ki-67 and the combined expression levels of the three enzymes (p = 0.002; Table 3). The results showed that in 68% of the patients with low expression of all three enzymes, there was no expression of ki-67, which indicated that there is only a low level of tumor cell proliferation in these patients. When at least one of the three histone-modifying enzymes showed high expression, we observed an increase in the percentage of ki-67 positive tumors (up to 56%), indicating more proliferation of the tumor cells in these patients. In summary, there are correlations between the combined expression levels of LSD1, HDAC2 and SIRT1 and tumor differentiation and between the combined expression levels of these enzymes and tumor cell proliferation.