High Expression of H3K9me3 Is a Strong Predictor of Poor Survival in Patients With Salivary Adenoid Cystic Carcinoma

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1 High Expression of H3K9me3 Is a Strong Predictor of Poor Survival in Patients With Salivary Adenoid Cystic Carcinoma Ronghui Xia, DDS; Rongrui Zhou, DDS, MDS; Zhen Tian, DDS, MDS, PhD; Chunye Zhang, DDS, MDS; Lizhen Wang, DDS, MDS; Yuhua Hu, DDS, MDS; Jing Han, DDS, MDS; Jiang Li, DDS, MDS, PhD Context. Histone methylation and acetylation play important roles in the carcinogenesis and progression of cancer. Objective. To investigate whether histone modifications influence the prognosis of patients with salivary adenoid cystic carcinoma (ACC). Design. The expression of histone H3 lysine 9 trimethylation (H3K9me3) and histone H3 lysine 9 acetylation (H3K9Ac) was assessed by immunohistochemistry in 66 specimens of primary ACC. Tests were used to determine the presence of any correlation between H3K9me3 and H3K9Ac levels and clinicopathologic parameters. Log-rank test and Cox proportional hazards regression models were used to analyze the survival data. Results. H3K9me3 expression was positively correlated with solid pattern tumors (P ¼.002) and distant metastasis (P ¼.001). Solid pattern tumors had lower H3K9Ac expression levels than cribriform-tubular pattern tumors (P ¼.03). Patients whose tumors showed high H3K9me3 expression and a solid pattern had a significantly poorer overall survival (OS) (P,.001 and P,.001, respectively) and disease-free survival (P,.001 and P ¼.01, respectively). Low H3K9Ac expression was correlated with poor OS (P ¼.05). The multivariate analysis indicated that high levels of H3K9me3 expression and solid pattern tumors were independent prognostic factors that significantly influenced OS (P ¼.004 and P ¼.04, respectively). H3K9me3 expression was identified as the only independent predictor of disease-free survival (P ¼.006). Conclusions. Our results suggest that high levels of H3K9me3 expression are predictive of rapid cell proliferation and distant metastasis in ACC. Compared with histologic patterns, H3K9me3 might be a better predictive biomarker for the prognosis of patients with salivary ACC. (Arch Pathol Lab Med. 2013;137: ; doi: /arpa OA) Salivary adenoid cystic carcinoma (ACC) is one of the most common malignancies of salivary glands in eastern China populations, comprising 9.75% of all salivary gland neoplasms and 30.42% of all salivary gland malignancies. 1 Patients with ACC have a relatively high 5-year survival rate, but their longer-term prognosis is less optimistic as a result of tumor recurrence after treatment and distant metastasis to sites that include the lung, bone, and liver. 2,3 The precise evaluation of the prognosis of Accepted for publication April 6, Supplemental digital content is available for this article at www. archivesofpathology.org in the December 2013 Table of Contents. From the Department of Oral Pathology, 9th People s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai, China (Drs Xia, Tian, Zhang, Wang, Hu, Han, and Li); the Department of Oncology and Diagnostic Sciences, Dental School, University of Maryland, Baltimore, Maryland (Dr Xia); and the Department of Oral Medicine, Shanghai Stomatological Disease Center, Shanghai, China (Dr Zhou). Ronghui Xia and Rongrui Zhou contributed equally to this work. The authors have no relevant financial interest in the products or companies described in this article. Reprints:JiangLi,DDS,MDS,PhD,DepartmentofOralPathology, 9th People s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai , China ( lijiang182000@yahoo.com). patients with ACC might help to determine the most appropriate treatment strategies, thereby improving their survival and quality of life. Therefore, the discovery of accurate prognostic biomarkers is an important area of ACC research. In addition to genetic alterations, epigenetic modifications contribute to the development and progression of many malignant tumors. 4 6 Alterations in DNA methylation, histone modifications, and microrna expression 7,8 can change gene expression without changing the DNA sequence. Epigenetic alterations may influence several aspects of tumor cell biology, including cell proliferation, differentiation, and apoptosis, and might be linked to the clinical outcomes of patients. 9 Many studies have focused on aberrant DNA methylation in cancer, and our previous studies demonstrated that hypermethylation of the promoter regions of CDKN2A, RASSF1A, and DAPK commonly occurs in ACC; furthermore, E-cadherin is frequently inactivated in ACC tumors through promoter hypermethylation. 10,11 In comparison to DNA methylation, histone modifications are less well characterized in ACC. However, histone modifications appear to play important roles in organizing the nuclear architecture, with consequent effects on the regulation of gene transcription. 4,12 Histone H3 lysine 9 trimethylation (H3K9me3) is a histone modification that Arch Pathol Lab Med Vol 137, December 2013 H3K9me3 Expression for Predicting Survival in ACC Xia et al 1761

2 Figure 1. Representative photomicrographs of immunohistochemical staining for histone H3 lysine 9 trimethylation (H3K9me3). A, High expression of H3K9me3 in a cribriform-tubular pattern tumor. B, Low expression of H3K9me3 in a cribriform-tubular pattern tumor. C, High expression of H3K9me3 in a solid pattern tumor. D, Low expression of H3K9me3 in a solid pattern tumor (original magnifications 340 [A, B, and C], 320 [D], and 380 [insets B and D]). occurs in the N-terminal of lysine residues. It has been correlated with tumor suppressor gene silencing in colorectal cancer, 13 hepatocellular cancer, 14 and breast cancer. 15 Histone acetylation is an important epigenetic mechanism that regulates gene transcription through altering the chromatin structure. 16 Specific marks, such as lysine acetylation (H3K9Ac, H3K18Ac, and H4K12Ac), are associated with activating gene promoters. 17,18 The prognostic value of histone modifications remains controversial. High levels of H3K9me3 have been correlated with a poor survival rate in gastric adenocarcinoma, 19 but a decrease in H3K9me3 expression is associated with a poor prognosis of patients with non small cell lung cancer. Low expression levels of H3K9Ac have also been correlated with poor survival outcomes in non small cell lung cancer. 20 Together, H3K9me3 and H3K9Ac expression could be potential prognostic biomarkers for malignant tumors. However, whether the expression of H3K9me3 and H3K9Ac is correlated with prognosis in patients with ACC is not known. Therefore, we determined H3K9me3 and H3K9Ac expression levels in 66 ACC biopsy specimens in a Chinese population and correlated these histone modifications with both clinicopathologic parameters and patient outcomes. MATERIALS AND METHODS Patients Tumor samples of 66 patients with ACC were included in the present study. Patients underwent radical surgery without either preoperative chemotherapy or radiotherapy at the Department of Oral and Maxillofacial Surgery, 9th People s Hospital, Shanghai Jiao Tong University (Shanghai, China) between 1999 and At 3 to 4 weeks postoperatively, adjuvant radiotherapy was administered to 51 patients with megavoltage equipment using cobalt 60. For patients with positive surgical margins, a radiation dosage of 66 Gy was given, compared to 60 Gy for patients with negative surgical margins. Thirteen patients received concomitant chemotherapy using a combination of either cisplatin and 5-fluorouracil or cisplatin, 5-fluorouracil, and adriamycin. Clinicopathologic parameters, including patient age, sex, tumor site, histologic pattern, perineural invasion, surgical margin status, TNM stage at the time of surgery, and follow-up information, such as the presence of recurrence and distant metastasis, were obtained from pathologic reports and medical records. According to the histologic typing of salivary gland tumors by the World Health Organization, 21 ACC is classified into cribriform, tubular, and solid patterns Arch Pathol Lab Med Vol 137, December 2013 H3K9me3 Expression for Predicting Survival in ACC Xia et al

3 Figure 2. Representative photomicrographs of immunohistochemical staining for histone H3 lysine 9 acetylation (H3K9Ac). A, High expression of H3K9Ac in a cribriform-tubular pattern tumor. B, Low expression of H3K9Ac in a cribriform-tubular pattern tumor. C, High expression of H3K9Ac in a solid pattern tumor. D, No expression of H3K9Ac in a cribriform-tubular pattern tumor (original magnifications 340 [A, B, and D], 320 [C], and 380 [inset C]). The histologic pattern was determined in this study according to the following criteria cribriform-tubular pattern: no solid component or solid component less than 30%; solid pattern: solid component of 30% or greater. The TNM stage of each case was determined according to the American Joint Committee on Cancer staging system. 22 The study was approved by the Shanghai 9th People s Hospital Ethics Committee and informed consent was obtained from each patient. Immunohistochemistry We used specific rabbit polyclonal antibodies (all from Abcam Inc, Cambridge, United Kingdom) to detect H3K9me3 and H3K9Ac expression in ACC specimens. The appropriate dilutions and incubation times had been previously determined. After deparaffinization through a graded series of xylene, the sections were rehydrated in a graded series of alcohol. Heat-mediated antigen retrieval using 0.01 M sodium citrate buffer (ph 6.0) was performed and endogenous peroxidase was quenched with 3% hydrogen peroxide for 20 minutes at room temperature. The sections were then incubated with normal goat serum to reduce nonspecific binding. The sections were incubated with rabbit polyclonal H3K9me3 antibody (1:300) and H3K9Ac antibody (1:200) at 48C overnight. Detection was accomplished with the Dako EnVision System (Carpinteria, California), and the expression of both H3K9me3 and H3K9Ac was detected with the diaminobenzidine chromogen system (Dako). The sections were counterstained with hematoxylin and then dehydrated and mounted. Blank controls were whole-tissue sections stained in the absence of the primary antibody. Evaluation of Immunohistochemical Staining All the immunohistochemical staining was evaluated by 2 independent pathologists (W.L. and H.Y.) who were blinded to the clinical data. In the case of discordance, both observers defined the final score of the sample through discussion. The score of each section was based on the percentage and intensity of positively stained cells. The percentage score was as follows: no positive cells (0), fewer than 25% positive tumor cells (1), 25% to 50% positive tumor cells (2), 51% to 75% positive tumor cells (3), and greater than 75% positive tumor cells (4). The intensity score was as follows: no staining (0), light yellow (1), yellow-brown (2), and dark brown (3). The sections were graded into 2 groups by multiplying the percentage and intensity scores, as follows: low expression (score,6) and high expression (score 6). Statistical Analysis Statistical analysis was performed with SPSS 17.0 software (SPSS Inc, Chicago, Illinois). The v 2 test was used to determine the relationship between the expression of H3K9me3 or H3K9Ac and Arch Pathol Lab Med Vol 137, December 2013 H3K9me3 Expression for Predicting Survival in ACC Xia et al 1763

4 Characteristics Table 1. Correlation Between H3K9me3 and H3K9Ac Expression and Clinicopathologic Parameters of Adenoid Cystic Carcinoma a H3K9me3 Expression H3K9Ac Expression P Low High Value Low High Age, y Sex Male Female Tumor site Minor glands Major glands Histologic pattern Cribriform-tubular Solid Perineural invasion Yes No T stage T1þT T3þT N stage Nþ N M stage M M Recurrence Yes No Distant metastasis Yes No Abbreviations: H3K9Ac, histone H3 lysine 9 acetylation; H3K9me3, histone H3 lysine 9 trimethylation; Nþ, positive lymph node; N, negative lymph node. a Bolded values are statistically significant (P.05). P Value clinicopathologic parameters. Survival curves were estimated by using the Kaplan-Meier method, and the significance of differences in the overall survival (OS) and disease-free survival (DFS) rates was tested by the log-rank test. The variables with a significant difference (P.05) according to the log-rank test were subjected to a multivariate analysis by using the Cox proportional hazards regression model. The Enter method was then used to determine a final Cox model. All of the tests were 2-sided and P.05 was considered to be statistically significant. RESULTS Clinicopathologic Characteristics Detailed clinicopathologic characteristics and follow-up information for each patient are presented in the Supplementary S1 file (see supplemental material file at in the December 2013 table of contents). Of the 66 patients with ACC, 36 (54.55%) were men and 30 (45.45%) were women, ranging in age from 16 to 82 years (mean, years). Twenty-seven tumors (40.91%) originated from the major salivary glands and 39 tumors (59.09%) originated from the minor salivary glands. Histologically, 49 cases (74.24%) had a cribriform-tubular pattern and 17 (25.76%) had a solid pattern. Two samples were excluded from the analysis of the correlation between distant metastasis and survival as the disease had metastasized to the lung before surgery in these patients. Perineural invasion, positive surgical margins, local recurrence, and distant metastasis were observed in 50 (75.76%), 21 (31.82%), 21 (31.82%), and 34 (51.52%) patients, respectively. The mean follow-up duration was months (range, months). Correlation Between H3K9me3, H3K9Ac Expression, and Clinicopathologic Parameters The expression of H3K9me3 (Figure 1A, 1B, 1C, and 1D) and H3K9Ac (Figure 2A, 2B, 2C, and 2D) was manifested as nuclear staining. The correlations between the expression levels of H3K9me3 and H3K9Ac expression with clinicopathologic parameters are presented in Table 1. In particular, tumors with a solid pattern had higher levels of H3K9me3 expression than those with a cribriform-tubular pattern (P ¼.002), while tumors with a cribriform-tubular pattern had higher levels of H3K9Ac than solid pattern tumors (P ¼.03) (Figure 3A, 3B, 3C, and 3D). As adenoid cystic carcinoma is a biphasic tumor that consists of 2 main cell types, myoepithelial-like cells and ductal cells, we analyzed whether H3K9me3 and H3K9Ac expression patterns correlated to the tumor cell types. We found that myoepithelial cells and ductal cells did not represent different H3K9me3 and H3K9Ac expression patterns. Of the 34 patients who had distant metastasis, 26 (76.5%) had high levels of H3K9me3, whereas only 11 of 32 patients (34.4%) who had no distant metastasis had high levels of H3K9me3 expression; high levels of H3K9me3 expression therefore correlated with distant metastasis (P ¼.001). There was no statistically significant correlation between the 1764 Arch Pathol Lab Med Vol 137, December 2013 H3K9me3 Expression for Predicting Survival in ACC Xia et al

5 Figure 3. Histone H3 lysine 9 trimethylation (H3K9me3) and histone H3 lysine 9 acetylation (H3K9Ac) expression was correlated with the histologic pattern of salivary adenoid cystic carcinoma. A, High expression of H3K9Ac in a cribriform-tubular pattern tumor. B, Low expression of H3K9me3 in a cribriform-tubular pattern tumor. C, Low expression of H3K9Ac in a solid pattern tumor. D, High expression of H3K9me3 in a solid pattern tumor (original magnifications 320 [A and B] and 340 [C, D, and inset A]). expression level of H3K9me3 or H3K9Ac and other clinicopathologic parameters. Correlation Between H3K9me3, H3K9Ac Expression, and Survival Patients whose tumors had high levels of H3K9me3 showed significantly poorer OS outcomes than those with low levels of H3K9me3 (P,.001) (Figure 4, A), while patients with high H3K9Ac expression had a significantly better OS than those with low expression levels (P ¼.05) (Figure 4, B). In terms of clinicopathologic variables, solid pattern tumors, M1 stage tumors, and distant metastasis were correlated with worse OS (P,.001, P ¼.004, and P ¼.001, respectively) (Figure 4, C through E). We also found that high H3K9me3 expression, solid pattern tumors, and positive surgical margins were correlated with poorer DFS rates (P,.001, P ¼.01, and P ¼.03, respectively) (Figure 4, F through H). We then determined whether H3K9me3, H3K9Ac expression, and clinicopathologic parameters were correlated with patient outcomes by using the Cox proportional hazards regression model. In the univariate analysis, high H3K9me3 expression levels, solid pattern tumors, and M1 stage were associated with poor OS, and the H3K9me3 expression was the most striking factor. H3K9me3 expression, histologic pattern, and margin status were associated with poor DFS (Figure 5). To determine which variables were independent survival predictors, a multivariate analysis was performed and showed that H3K9me3 expression (P ¼.004) and histologic pattern (P ¼.04) were independent factors for the estimation of OS. H3K9me3 expression (P ¼.006) was the only independent factor for the estimation of DFS (Table 2). COMMENT Histones are subject to posttranslational covalent modifications, such as methylation and acetylation. These modifications can control the activity of the surrounding DNA. 23 The cellular levels of histone modifications, based on tissue specimens, are related to cancer prognosis in different tumor types. 24 Although the information about the histone alterations in ACC tumorigenesis and prognosis was limited, some previous studies 10,25 have reported that CDKN2A might be inactivated by promoter hypermethyla- Arch Pathol Lab Med Vol 137, December 2013 H3K9me3 Expression for Predicting Survival in ACC Xia et al 1765

6 Figure 4. A through E, Kaplan-Meier survival analyses of the relationship between histone H3 lysine 9 trimethylation (H3K9me3) expression (A), histone H3 lysine 9 acetylation (H3K9Ac) expression (B), histologic pattern (C), M stage (D), and distant metastasis (E) with overall survival. F through H, Kaplan-Meier survival analyses of the relationship between H3K9me3 (F), histologic pattern (G), and margin status (H) with disease-free survival. Abbreviation: c/t, cribriform/tubular. tion and loss of heterozygosity in ACC. CDKN2A is known to be an important tumor suppressor gene and one recent study 26 showed that CDKN2A could be reactivated by decreasing histone H3K9 methylation and increasing histone H3K9 acetylation level. Therefore, H3K9me3 and H3K9Ac might influence CDKN2A expression in ACC and hence the prognosis of the patients. In the current study, we used immunohistochemistry to evaluate H3K9me3 and H3K9Ac expression levels in ACC tissue specimens. High H3K9me3 and low H3K9Ac expression levels correlated with the solid tumor growth pattern of ACC, in which the tumor cells have a higher proliferative activity and are usually more aggressive. This could be because H3K9me3 and H3K9Ac might regulate the expression of cell proliferation related genes, such as Cip1/p21, 26 ERK, 27 and Notch1. 28 Another phenomenon in this study was that myoepithelial and ductal tumor cells of ACC did not reveal different H3K9me3 and H3K9Ac expression patterns. We proposed 1766 Arch Pathol Lab Med Vol 137, December 2013 H3K9me3 Expression for Predicting Survival in ACC Xia et al

7 Figure 5. Summary of the univariate analysis for overall survival and disease-free survival for patients with salivary adenoid cystic carcinoma. The forest plot shows that patients with solid pattern tumors, M1 stage tumors, and higher levels of histone H3 lysine 9 trimethylation (H3K9me3) expression had a poorer overall survival. Patients with a solid pattern tumor, positive margins, and higher levels of histone H3K9me3 expression had a poorer disease-free survival. : Statistically significant values (P.05). Abbreviations: CI, confidence interval; CT, chemotherapy; c/t, cribriform/ tubular pattern; HRs, hazard ratios; n, negative; H3K9Ac, histone H3 lysine 9 acetylation; H3K9me3, histone H3 lysine 9 trimethylation; p, positive; RT, radiation therapy; s, solid pattern. that although myoepithelial and ductal cells represented different phenotypes, they probably still had a similar genotype since both of the cells originated from intercalated duct reserve cells of the salivary gland. We also found that high levels of H3K9me3 expression were significantly correlated with distant metastasis, which may be because H3K9me3 can suppress the expression of cell invasion and distant metastasis related genes, such as E-cadherin, 29 BRMS1, 30 and CXCR4. 31 The prognostic value of H3K9me3 and H3K9Ac expression has been established in gastric adenocarcinoma, 19 acute myeloid leukemia, 32 lung cancer, 20 and ovarian epithelial Table 2. Multivariate Analysis Using Cox Proportional Hazards Regression Models to Estimate Overall Survival (OS) and Disease-Free Survival (DFS) a Variables P Value Hazard Ratio 95% CI OS analysis Histologic pattern (c/t versus s) M stage (M0 versus M1) H3K9me3 expression (low versus high) DFS analysis Histologic pattern (c/t versus s) Margin status (negative versus positive) H3K9me3 expression (low versus high) Abbreviations: CI, confidence interval; c/t, cribriform/tubular pattern; H3K9me3, histone H3 lysine 9 trimethylation; s, solid pattern. a Bolded values are statistically significant (P.05). tumors, 33 although it remains controversial. For example, high H3K9me3 expression levels have been correlated with poor prognosis in gastric adenocarcinoma, but decreased levels of H3K9me3 and H3K9Ac expression have been associated with poor prognosis in lung cancer. In the current study, we used log-rank test and Cox proportional hazards regression models to analyze the survival data. We found that high H3K9me3 expression indicated poor OS and DFS rates for patients with ACC. These findings were in accordance with the study by Park et al, 19 which showed that stronger H3K9me3 expression corresponded to a worse prognosis in gastric adenocarcinoma. Thus, the prognostic value of H3K9me3 expression might be different in different tumors. We also found that low H3K9Ac expression correlated with poor survival in ACC. Pasini et al 34 reported that histone H3 lysine 27 trimethylation (H3K27me3) and acetylation (H3K27Ac) acted as an antagonistic switch in the transcriptional regulation of target genes. Therefore, to explain our current results, we hypothesized that the methylation and acetylation of H3K9 might have the same antagonistic switch function in suppressing the expression of tumor suppressor genes. This might help to explain why high H3K9me3 and low H3K9Ac expression were correlated with poor survival. We then analyzed whether the H3K9me3, H3K9Ac, and clinicopathologic parameters were risk factors for ACC. Although the univariate analysis showed that H3K9me3 expression, histologic pattern, and M stage were risk factors for predicting OS, and H3K9me3 expression, histologic pattern, and margin status could predict DFS, the multivariate analysis indicated that only H3K9me3 expression and histologic pattern could act as independent factors for estimating the OS. More importantly, H3K9me3 expression was the only independent factor that could estimate the DFS. This might have been because histone modifications Arch Pathol Lab Med Vol 137, December 2013 H3K9me3 Expression for Predicting Survival in ACC Xia et al 1767

8 can regulate the expression of certain tumor suppressor genes, such as p16, RIZ1, 14 and BRCA1, 35 which leads to the inhibition of tumor cell apoptosis. In addition, histone modifications can also regulate tumor suppressor genes that are associated with cell proliferation and distant metastasis, as described above. Cell growth dysregulation, and the inhibition of apoptosis and distant metastasis, are features that might correlate with poor prognosis in some tumors. Therefore, we hypothesized that high levels of H3K9me3 expression increased tumor cell growth, invasion, and distant metastasis, which result in a poor prognosis in ACC. With regard to clinicopathologic parameters, with the exception of the histologic pattern, our results revealed that sex, perineural invasion, tumor site, recurrence, and postoperative radiotherapy and chemotherapy had no significant prognostic value in predicting either OS or DFS. Postoperative radiation therapy for ACC was usually performed in most institutions. Recent studies 36,37 have indicated that surgery combined with postoperative treatment could achieve better local control and improve disease-free survival. However, the role of chemotherapy in ACC is controversial and no definitive protocols have become universally accepted. 3 In the current study, our analysis did not show significant relationship between radiation/chemotherapy and OS and DFS, which could be for 2 reasons. First, for radiation therapy, in other research studies, 35,36 which showed that radiation therapy had a positive effect on patients survival, almost every patient received postoperative radiation therapy, but in our cohort, only 77.3% (51 of 66) received postoperative radiation therapy. Second, for the chemotherapy, in the current study, only 19.7% (13 of 66) of patients underwent postoperative treatment. The lower adjuvant therapy rate in our institution may explain why radiotherapy/chemotherapy was not correlated with OS and DFS. One limitation of this study was the fact that analyzing histone modifications via immunohistochemistry cannot provide information regarding changes in the activity of specific tumor suppressor genes. Future studies will be needed to address whether H3K9me3 and H3K9Ac expression changes could lead to the silencing of specific tumor suppressor genes in ACC. Nevertheless, our current research detected the expression level of H3K9me3 and H3K9Ac in 66 ACC specimens and demonstrated that an assessment of those histone modifications, especially H3K9me3, might be useful for predicting the prognosis of patients with ACC. CONCLUSION Our findings highlighted that H3K9me3 and H3K9Ac expression was correlated with histologic pattern and the presence of distant metastasis in ACC. 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