DAXX/ATRX and MEN1 genes are strong prognostic markers in pancreatic neuroendocrine tumors.

BACKGROUND
PanNETs shows heterogeneous biological behaviors. The aim was to investigate prognostic markers based on most frequently mutated genes in PanNETs.


RESULTS
There was a total of 76 patients (M: 39, F: 37) with pathologically proven PanNETs. ATRX/DAXX and MEN1 protein expression was detected in 16 (21%) and 31 (41%) patients, respectively. The mean OS of the total study patients was 16 years, and DFS was 17 years among the 68 patients with curative resections. PanNETs presented with distant metastasis or loss of ATRX/DAXX protein expression was the independent prognostic factors associated with poor OS. In curative resected PanNETs, there was no significant difference in the mean DFS according to ATRX/DAXX or MEN1 protein. However, there was statistically significant difference in survival after the recurrence according to the expression of ATRX/DAXX protein; Y/N: 10 vs. 15 years, p < 0.001. In metastatic PanNETs, we could find out OS was significantly longer in negative protein expression of ATRX/DAXX and MEN1 groups; 7 vs. 1 years, p < 0.001, 6 vs. 2 years, p = 0.02, respectively.


MATERIALS AND METHODS
The histologically proven PanNETs were enrolled and the clinicopathologic and genetic alterations were evaluated.


CONCLUSIONS
Protein expression of MEN1 and DAXX/ATRX can be prognostic markers for PanNETs. Further investigation in genetic alterations of PanNETs may give us insights understanding the behavior of PanNETs.


INTRODUCTION
Pancreatic neuroendocrine tumors (PanNETs) are rare neoplasms which have an incidence of approximately one per 100,000 individuals per year and represent 3% of all pancreatic tumors [1,2].PanNET is an important form of pancreatic neoplasia and the 10-year survival rate is 40% [3,4].Several studies have documented a trend towards the increasing incidence and prevalence of PanNETs [5].Although usually indolent, the biological behavior of PanNET ranges varies from benign to malignant ones, and some PanNETs may also show very aggressive behavior with rapid progression and poor prognosis [6][7][8][9][10][11].Predictors of prognosis include nonfunctioning tumor, tumor size, the presence and the site of metastases, the degree of tumor differentiation, Ki-67 and spontaneous tumor growth rapidity [12][13][14][15][16].However, the prognostic factors of PanNET are still debatable because of their rarity and heterogeneity of biologic and clinical features.

Research Paper www.impactjournals.com/oncotarget
Recently, Jiao et al. and Marinoni et al. both conducted the large-scale mutational analysis with the help of high throughput techniques in patients with PanNETs so far.First of all, Jiao et al. reported that the gene mutations in MEN1 and DAXX (death-domain-associated protein)/ ATRX (α thalassemia/mental retardation syndrome X-linked) genes emerged as the most frequent molecular events and associated with better prognosis [17].On the other hands, Marinoni et al. reported that the loss of DAXX and ATRX are related with chromosome instability and poor survival of patients with PanNETs which seemed to contradictory to the results of Jiao et al. study [17,18].The aims of this study were to identify the genetic alterations which enable us to predict the prognosis and further more survival of the patients with PanNETs.

Characteristics of the study patients
The baseline characteristics were described in Table 1.The total of 76 patients with pathologically proven PanNETs was enrolled in tertiary, teaching hospital.There were 39 males (51%) and the median age of study patients was 54 years old (range 21 to 76 years).There were 71 patients (93%) who underwent operation including palliative surgery and 68 patients (96%) had curative resections; 51 patients in stage IA,B, 16 in IIA,B and 1 patient who had resection for pancreas mass and single solitary metastasis resection from the liver.Among the resected PanNETs, there were 4 functioning tumors; 2 insulinomas, 1 gastrinoma, and 1 somatostatinoma.The median size of tumor was 23mm ranged from 3 to 200 mm and the followings is the location of PanNETs; head & uncinated: 40, body & tail: 34 and multifocal: 2. In addition, there were 9 (12%) patients who were presented with metastatic PanNETs; AJCC stage IA, IB: 51, IIA, IIB: 16, III:0, IV 9 patients.Among the 68 resected PanNETs, 7 (10%) patients had recurrences after the curative resection.

Pathologic characteristics
All study patients were pathologically proven as PanNETs and the grading system from 2010 WHO classification of neuroendocrine tumors was used for the study patients; G1: 53 (70%), G2: 20 (26%) and G3: 3 (4%).IHC staining results with ATRX/DAXX and MEN1 are shown in Figure 1 and Table 1.In Figure 1, panel A-C showed the positive IHC staining result of DAXX/ ATRX and MEN1 which indicated positive expression for DAXX/ATRX and MEN1 proteins.Panel D-I showed negative staining results due to the loss of ATRX/DAXX and MEN1 protein expression.Among the study patients, positive expression for ATRX/DAXX and MEN1 protein were detected in 16 (21%) and 31 (41%) patients, respectively.There were 41 (54%) patients who had both negative expressions for ATRX/DAXX and MEN1 proteins.

Overall survival and disease-free survival of the study patients
As we mentioned above, there was a total of 76 study patients and 68 patients who underwent curative resection among them.The mean overall survival (OS) was 15.5 years (95 % CI 13.8-17.1 years) and 16 out of 76 study patients (21%) died during the follow-up (Figure 2A).In addition, DFS was 16.5 years (ranged from 14.9 to 18.1years) among the 68 patients with the curative resections (Figure 2B).

Prognostic factors affecting OS of the study patients
The following clinical and histological parameters associated with OS were analyzed: age, staging at the time of diagnosis, lymph node status, curative intent surgery, WHO grade and protein expression status of ATRX/DAXX and MEN1.Among them, distant metastasis (mean OS 3.0 years vs. 16.9 years, p < 0.001), lymph node positive (mean OS 8.8 years vs. 16.0 years p = 0.025), curative intent surgery (mean OS 16.7 years vs. 2.9 years, p < 0.001), WHO grade (mean OS G1 16.4 years vs. G2 15.4 years vs. G3 0.6 years, p < 0.001), and negative expression for ATRX/DAXX protein (mean OS 15.3 years vs. 10.8 years, p < 0.001) were significant prognostic factors associated with OS in univariate analysis (Figure 3A-3E).On the other hands, MEN1 protein expression status did not have statistically significant difference in OS (mean OS 16.4 years vs. 14.0 years, p = 0.08).In multivariate analysis, patients presented with positive expression of ATRX/DAXX protein (HR 3.809, 95% CI 1.064-13.630,p = 0.04) was the only independent prognostic factors associated with poor OS (Table 2).

Subgroup analysis in curatively resected PanNETs vs. metastatic PanNETs
There was no significant difference in the mean disease-free survival according to ATRX/DAXX (Y/N: 17.1 years vs. 15.4 years, p = 0.77) or MEN1 protein expression status (Y/N: 16.5 years vs. 16.0 years, p = 0.47) in curatively resected PanNETs (Figure 4A).However, among the patients with curatively resected PanNETs, both negative protein expression tumors seemed to have longer DFS which was opposite to the result that negative ATRX/DAXX protein expression was the independent prognostic factor for longer OS in the study patients.Therefore, we also evaluated unique parameter such as survival after the recurrence since PanNETs have significantly longer survival compared to PDACs.There was clearly statistically significant difference in www.impactjournals.com/oncotargetsurvival after the recurrence according to ATRX/DAXX protein expression status; Y/N: 9.7 years vs. 15.3 years, p < 0.001 in Figure 4B.On the other hand, MEN1 protein expression status did not make significant difference in survival after the recurrence; Y/N: 12.4 years vs. 16.4 years, p = 0.08.However, there was a still tendency to have longer survival after the recurrence in patients with negative MEN1 protein expression.
In metastatic PanNETs, we could find out OS was significantly longer in negative ATRX/DAXX and MEN1 protein groups; Figure 5A

DISCUSSION
Hallmarks of PanNETs are having heterogeneous and wide spectrum of clinical paths, however, there is absence of strong prognostic markers for recurrences [19].The investigation and managements needs to be individualized for each patient and therefore, finding of surrogate markers to predict its prognosis can be very important [20].The most frequently mutated genes have been reported with the help of high throughput techniques and they are the followings; MEN1 and DAXX/ATRX genes.Somatic mutations of MEN1 and DAXX/ATRX genes were inactivating mutations [17,[21][22][23][24]. DAXX/ ATRX genes specify proteins implicated in chromatin remodeling.MEN1 gene encodes menin, a histone methyltransferase complex and acts as a tumor suppressor.DAXX/ATRX genes, either of the two subunits consists of a transcription/chromatin remodeling complex [17,[21][22][23][24].To explore and investigate the prognostic markers of PanNETs, we have investigated 76 patients with pathologically proven PanNETs based on genetic alterations and had relatively thorough and longer followup data compared to the previous studies [17,18].ATRX/ DAXX (either one of the genes since they are mutually exclusive) and MEN1 protein expression were detected in 16 (21%) and 31 (41%) patients, respectively.MLL/SET1-like histone methyltransferase complex and regulates chromatin remodeling, functioning as activator or suppressor of gene transcription according to the cell type [25].For example, menin acts as a tumor activator in promoting MLL-dependent leukemias, but acts as a tumor suppressor in neuroendocrine tumors [25].As we have showed in Figure 4A, patients with positive for MEN1 protein expression had significantly longer DFS compared to negative for MEN1 protein expression group.DAXX mutation decreases p53 levels, diminishing the check point for cellular/DNA damages [25].In addition, changes in the nucleotide sequence often resulted in nonsense mutations that are generally relevant to tumor suppressor genes [25].Furthermore, Heaphy et al. reported a perfect correlation between the loss of ATRX or DAXX function and the presence of a telomerase-independent telomere maintenance mechanism known as alternative lengthening of telomeres (ALT) [26].The association of ATRX and DAXX inactivation with the ALT phenotype might explain previous observations in other tumor types that connected the ALT phenotype with improved prognosis [27,28].
Thompson et al. also reported that the resulting ALT phenotype is the basis for the good prognosis; probably by preventing the initiation of widespread chromosomal ).We defined negative expression if the pattern was that of cytoplasmic accumulation with nuclear clearing, as long as adequate internal controls were present.When differences between the observers occurred, the slides were reinvestigated jointly by both investigators and then determined.Representative pictures of nuclear (Nu), cytoplasmic staining (Cy) and negative (N) staining of three markers were displayed in Figure 1.MEN1 was positively stained in nuclei of non-neoplastic acinar cells which were used in internal positive control (Supplementary Figure 1A).ATRX was positively stained in macrophages or lymphocytes which were used in internal positive control (Supplementary Figure 1B).DAXX was positively stained in cytoplasm of non-neoplastic ductal epithelial cells which were used in internal positive control (Supplementary Figure 1C).Positive and negative controls were included with each staining procedure to ensure consistency between consecutive runs.

Statistical analysis
The overall survival and disease-free survival were analyzed by Kaplan-Meier method, and the significance of differences was determined by the log rank test.Multivariate analysis was performed by Cox proportional hazard regression modeling.A P value of < 0.05 was considered to be statistically important.Statistical analysis was conducted by using the IBM SPSS Statistics version 19.0 (SPSS Inc, Chicago, Il., USA).