DAXX/ATRX, MEN1, and mTOR Pathway Genes Are Frequently Altered in Pancreatic Neuroendocrine Tumors

See allHide authors and affiliations

Science  04 Mar 2011:
Vol. 331, Issue 6021, pp. 1199-1203
DOI: 10.1126/science.1200609


Pancreatic neuroendocrine tumors (PanNETs) are a rare but clinically important form of pancreatic neoplasia. To explore the genetic basis of PanNETs, we determined the exomic sequences of 10 nonfamilial PanNETs and then screened the most commonly mutated genes in 58 additional PanNETs. The most frequently mutated genes specify proteins implicated in chromatin remodeling: 44% of the tumors had somatic inactivating mutations in MEN1, which encodes menin, a component of a histone methyltransferase complex, and 43% had mutations in genes encoding either of the two subunits of a transcription/chromatin remodeling complex consisting of DAXX (death-domain–associated protein) and ATRX (α thalassemia/mental retardation syndrome X-linked). Clinically, mutations in the MEN1 and DAXX/ATRX genes were associated with better prognosis. We also found mutations in genes in the mTOR (mammalian target of rapamycin) pathway in 14% of the tumors, a finding that could potentially be used to stratify patients for treatment with mTOR inhibitors.

Pancreatic neuroendocrine tumors (PanNETs) are the second-most common malignancy of the pancreas. The 10-year survival rate of patients with PanNETs is only 40% (13). PanNETs are usually sporadic but can arise in multiple endocrine neoplasia type 1 and more rarely in other syndromes, including von Hippel-Lindau (VHL) syndrome and tuberous sclerosis (4). “Functional” PanNETs secrete hormones that cause systemic effects, whereas “nonfunctional” PanNETs do not and therefore cannot always be readily distinguished from other neoplasms of the pancreas. Nonfunctional PanNETs grow silently, and patients often present with either an asymptomatic abdominal mass or symptoms of abdominal pain secondary to compression by a large tumor. Surgical resection is the treatment of choice, but many patients present with unresectable tumors or extensive metastatic disease, and medical therapies are relatively ineffective in these cases.

There is currently insufficient information about this tumor to either predict prognosis of patients diagnosed with PanNETs or to develop companion diagnostics and personalized treatments to improve disease management. Biallelic inactivation of the MEN1 gene, usually through a mutation in one allele coupled with loss of the remaining wild-type allele, occurs in 25 to 30% of PanNETs (5, 6). MEN1 is a tumor suppressor gene that, when mutated in the germline, predisposes to multiple endocrine neoplasia type 1 syndrome. Chromosomal gains and losses and expression analyses have revealed candidate loci for genes involved in the development of PanNETs, but these have not been substantiated through genetic or functional analyses (79).

To gain insights into the genetic basis of this tumor type, we determined the exomic sequence of ~18,000 protein-coding genes in a discovery set of 10 well-characterized sporadic PanNETs. A clinically homogeneous set of tumors of high neoplastic cellularity is essential for the successful identification of genes and pathways involved in any tumor type. Thus, we excluded small-cell and large-cell neuroendocrine carcinomas and studied only samples that were not part of a familial syndrome associated with PanNETs (table S1) (1). We microdissected tumor samples in order to achieve a neoplastic cellularity of >80%. DNA from the enriched neoplastic samples and from matched non-neoplastic tissue from 10 patients was used to prepare fragment libraries suitable for massively parallel sequencing. The coding sequences were enriched by capture with the SureSelect Enrichment System and sequenced by use of an Illumina GAIIx platform (10). The average coverage of each base in the targeted regions was 101-fold, and 94.8% of the bases were represented by at least 10 reads (table S2).

We identified 157 somatic mutations in 149 genes among the 10 tumors used in the discovery set. The mutations per tumor ranged from 8 to 23, with a mean of 16 (table S3). Of these mutations, 91% were validated by means of Sanger sequencing. There were some obvious differences between the genetic landscapes of PanNETs and those of pancreatic ductal adenocarcinomas (PDAC) (11). First, there were 60% fewer genes mutated per tumor in PanNETs than in PDACs. Second, the genes most commonly affected by mutation in PDACs (KRAS, TGF-β pathway, CDKN2A, and TP53) were rarely altered in PanNETs and vice versa (Table 1). Third, the spectrum of mutations in PDAC and PanNET were different, with C-to-T transitions more common in PDACs than in PanNETs and C-to-G transversions more common in PanNETs than in PDACs (table S4). This suggests that mutations in PanNETs and PDAC arise through different mechanisms, perhaps because of exposure to different environmental carcinogens or through the action of different DNA-repair pathways.

Table 1

Comparison of commonly mutated genes in PanNETs and PDAC based on 68 PanNETs and 114 PDACs.

View this table:

We next selected genes for further analysis that were well-documented components of a pathway that was genetically altered in more than one tumor because alterations in these genes are most likely to be clinically relevant. Four genes were mutated in at least two tumors in the discovery set: MEN1 in five, DAXX in three, PTEN in two, and TSC2 in two. ATRX was mutated in only one sample in the discovery set, but its product forms a heterodimer with DAXX and, therefore, is part of the same pathway, so it was also evaluated in the validation set. Similarly, PIK3CA was included because its product is part of the mammalian target of rapamycin (mTOR) pathway that includes PTEN and TSC2 (1214). The sequences of these genes were then determined by means of Sanger sequencing in a validation set consisting of 58 additional PanNETs and their corresponding normal tissues (Fig. 1, A and B). In total, somatic mutations in MEN1, DAXX, ATRX, PTEN, TSC2, and PIK3CA were identified in 44.1%, 25%, 17.6%, 7.3%, 8.8%, and 1.4% PanNETs, respectively (Table 2).

Fig. 1

(A and B) Examples of traces showing mutations in DNA isolated from cancer cells [(A) and (B), bottom] but not from normal cells of the same patient [(A) and (B), top]. (C and D) Kaplan-Meier plots of overall survival of patients with metastatic PanNETs. (C) Fifteen patients with a DAXX or ATRX gene mutation versus 12 patients in whom both genes were wild type (WT) [hazard ratio 0.22, 95% confidence interval (CI) 0.06 to 0.84, P = 0.03]. (D) Nine patients with mutations in MEN1 as well as either DAXX or ATRX versus six patients in which all three genes were WT (hazard ratio 0.07, 95% CI 0.009 to 0.53, P = 0.01). All types of mutations in these genes were included in the analysis.

Table 2

Mutations in MEN1, DAXX, ATRX, PTEN, TSC2, PIK3CA, and TP53 in human PanNETs. (hom) indicates these mutations appear homozygous.

View this table:

Of the 30 mutations in MEN1, 25 were inactivating mutations [18 insertions or deletions (“indels”), 5 nonsense, and 2 splice-site mutations], whereas five were missense. At least 11 were homozygous; in the others, the presence of “contaminating” DNA from normal cells made it difficult to reliably distinguish heterozygous from homozygous changes. MEN1 encodes menin, a nuclear protein that acts as a scaffold to regulate gene transcription by coordinating chromatin remodeling. It is an essential component of the MLL SET1–like histone methyltransferase (HMT) complex (1519). Overall, MEN1 was mutated in 30 of the 68 PanNETs used in the discovery and validation sets combined.

DAXX and ATRX were mutated in 17 and 12 PanNETs, respectively. No tumor with a mutation in DAXX had a mutation in ATRX, which is consistent with their presumptive function within the same pathway. Overall, 29 of 68 PanNETs (42.6%) had a mutation in this pathway. There were 11 indels and four nonsense mutations in DAXX and six indels and three nonsense mutations in ATRX. The three ATRX missense mutations were within the conserved helicase domain, whereas the DAXX missense mutations were nonconserved changes. Five DAXX and four ATRX mutations were homozygous, indicating loss of the other allele. The high ratio of inactivating-to-missense mutations in both genes establishes them as PanNET tumor suppressor genes. Loss of immunolabeling for DAXX and ATRX correlated with mutation of the respective gene (fig. S1, A and B, and table S5). From these data, we hypothesize that both copies of DAXX are generally inactivated, one through mutation and the other either through loss of the nonmutated allele or epigenetic silencing. We also hypothesize that both copies of ATRX are inactivated, one through mutation and the other through chromosome X inactivation. Recently, it has been shown that DAXX is an H3.3-specific histone chaperone (20). ATRX encodes for a protein that at the amino-terminus has an ADD (ATRX-DNMTT3-DNMT3L) domain and a carboxy-terminal helicase domain. Almost all missense disease-causing mutations are within these two domains (21). DAXX and ATRX interact, and both are required for H3.3 incorporation at the telomeres; ATRX is also required for suppression of telomeric repeat–containing RNA expression (2224). ATRX was recently shown to target CpG islands and G-rich tandem repeats (25), which exist close to telomeric regions.

We identified five PTEN mutations, two indels and three missense; six TSC2 mutations, one indel, one nonsense, and four missense; and one PIK3CA missense mutation. Previously published expression analyses have indicated that the expression of genes in the mTOR pathway is altered in most PanNETs (26, 27). Our data suggest that, at least at the genetic level, only a subset of PanNETs have alterations of this pathway. This finding may have direct clinical application through prioritization of patients for therapy with mTOR pathway inhibitors. Everolimus [also called Afinitor, RAD-001, and 40-O-(hydroxyethyl)-rapamycin] has been shown to increase progression-free survival in a subset of PanNET patients with advanced disease (28). If the mutational status of genes coding for proteins in the mTOR pathway predicts clinical response to mTOR inhibitors, it should be possible to select patients who would benefit most from an mTOR inhibitor through analysis of these genes in patients’ tumors (29, 30).

All 68 tumors evaluated in this study were from patients undergoing aggressive intervention (table S6) and included patients undergoing curative resection as well as those with metastatic disease. Mutations in MEN1, DAXX/ATRX, or the combination of both MEN1 and DAXX/ATRX were associated with prolonged survival relative to those patients whose tumors lacked these mutations (Fig. 1, C and D, and table S7). This was particularly evident in patients with metastatic disease and with mutations in both MEN1 and DAXX/ATRX: 100% of patients with PanNETs that had these mutations survived at least 10 years, whereas over 60% of the patients without these mutations died within 5 years of diagnosis (Fig. 1D). One possible explanation for the difference in survival is that mutations in MEN1 and DAXX/ATRX identify a biologically specific subgroup of PanNETs.

Whole-exome sequencing of pancreatic neuroendocrine tumors has led to the identification of previously unknown tumor suppressor genes and illuminated the genetic differences between the two major neoplasms of the pancreas. The mutations may serve to aid prognosis and provide a way to prioritize patients for therapy with mTOR inhibitors.

Supporting Online Material

Materials and Methods

Fig. S1

Tables S1 to S8


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank M. Whalen for expert technical assistance. This work was supported by a research grant from the Caring for Carcinoid Foundation, by the Lustgarten Foundation for Pancreatic Cancer Research, the Sol Goldman Pancreatic Cancer Research Center, the Joseph L. Rabinowitz Fund for Pancreatic Cancer Research, the Virginia and D. K. Ludwig Fund for Cancer Research, the Raymond and Beverly Sackler Research Foundation, the American Associatoion for Cancer Research Stand Up To Cancer–Dream Team Translational Cancer Research Grant, and by National Institutes of Health grants CA121113, P50CA062924, P01CA134292, and R01CA113669. N.P. B.V., L.D., V.E.V., and K.W.K. are members of the Scientific Advisory Board of Inostics, a company that is developing technologies for the molecular diagnosis of cancer. N.P. B.V., L.D., V.E.V., and K.W.K. are co-founders of Inostics and Personal Genome Diagnostics and are members of their Scientific Advisory Boards. The authors are entitled to a share of the royalties received by the university on sales of products related to genes described in this manuscript. N.P., B.V., K.W.K., L.A.D., and V.E.V. own Inostics and Personal Genome Diagnostics stock, which is subject to certain restrictions under university policy. The terms of these arrangements are managed by Johns Hopkins University in accordance with its conflict-of-interest policies. D.S.K. is a paid advisor for Novartis, which produces drugs for treatment of neuroendocrine tumors.

Stay Connected to Science

Navigate This Article