Gastroenterology 2014;146:453–460
CLINICAL—PANCREAS
Loss of DAXX and ATRX Are Associated With Chromosome
Instability and Reduced Survival of Patients With Pancreatic
Neuroendocrine Tumors
Ilaria Marinoni,1 Anja Schmitt Kurrer,1 Erik Vassella,1 Matthias Dettmer,1 Thomas Rudolph,1
Vanessa Banz,2 Fabio Hunger,1 Silvan Pasquinelli,1 Ernst–Jan Speel,3 and Aurel Perren1
1
Institute of Pathology, University of Bern, Bern, Switzerland; 2Department of Visceral Surgery and Medicine, Inselspital,
University Hospital Bern, Bern, Switzerland; 3Department of Molecular Cell Biology, Institute of Pathology University Medical
Center, Maastricht, Holland
See Covering the Cover synopsis on page 328.
BACKGROUND & AIMS: Sporadic pancreatic neuroendocrine
tumors (pNETs) are rare and genetically heterogeneous. Chro-
mosome instability (CIN) has been detected in pNETs from
patients with poor outcomes, but no specific genetic factors have
been associated with CIN. Mutations in death domain–associated
protein gene (DAXX) or ATR-X gene (ATRX) (which both encode
proteins involved in chromatin remodeling) have been detected in
40% of pNETs, in association with activation of alternative
lengthening of telomeres. We investigated whether loss of DAXX
or ATRX, and consequent alternative lengthening of telomeres, are
related to CIN in pNETs. We also assessed whether loss of DAXX
or ATRX is associated with specific phenotypes of pNETs.
METHODS: We collected well-differentiated primary pNET
samples from 142 patients at the University Hospital Zurich and
from 101 patients at the University Hospital Bern (both located
in Switzerland). Clinical follow-up data were obtained for 149
patients from general practitioners and tumor registries. The
tumors were reclassified into 3 groups according to the 2010
World Health Organization classification. Samples were analyzed
by immunohistochemistry and telomeric fluorescence in situ
hybridization. We correlated loss of DAXX, or ATRX, expression,
and activation of alternative lengthening of telomeres with data
from comparative genomic hybridization array studies, as well
as with clinical and pathological features of the tumors and
relapse and survival data. RESULTS: Loss of DAXX or ATRX
protein and alternative lengthening of telomeres were associ-
ated with CIN in pNETs. Furthermore, loss of DAXX or ATRX
correlated with tumor stage and metastasis, reduced time of
relapse-free survival, and decreased time of tumor-associated
survival. CONCLUSIONS: Loss of DAXX or ATRX is associated
with CIN in pNETs and shorter survival times of patients. These
results support the hypothesis that DAXX- and ATRX-negative
tumors are a more aggressive subtype of pNET, and could lead
to identification of strategies to target CIN in pancreatic tumors.
Keywords: Pancreas; Neoplasm ALT; Prognosis; Human.
P
ancreatic neuroendocrine tumors (pNETs) are rare
and comprise about 3% of all pancreatic tumors.
pNETs harbor a significant malignant potential; more than
50% of patients will die of their tumor within 10 years.1
Although surgery is the only curative option, the number
CLINICAL PANCREAS
of medical therapies available has increased over the past
10 years. All these therapies are able to induce stable dis-
ease in up to 30% of patients, and partial remissions in rare
instances.2–5 Today there is no means by which to select the
best therapy for an individual patient. Deeper understand-
ing of the molecular mechanisms leading to the develop-
ment of pNETs therefore is needed for a more personalized
treatment approach. Malignant pNETs are characterized by
chromosomal instability (CIN),6 but the causes of CIN in
pNETs are still elusive. Whole-exome sequencing recently
has shown very frequent somatic mutations in the death
domain–associated protein gene (DAXX) and ATR-X gene
(ATRX) in pNETs. Mutations in ATRX and DAXX were found
to be mutually exclusive, which suggests that the encoded
proteins function in the same pathway. Tumors with wild-
type ATRX and DAXX show intact nuclear protein expres-
sion, whereas this nuclear localization typically is lost in
tumors with mutations in DAXX or ATRX. DAXX- or ATRX-
mutated metastasized pNETs were shown to have a favor-
able outcome.7 However, too few patients have been
analyzed to date to be able to ascertain the impact of DAXX
and ATRX protein loss on disease outcome. DAXX or ATRX
loss has been found only in well-differentiated pNETs and
not in poorly differentiated pancreatic neuroendocrine
carcinomas, which instead show alterations in p53 and pRB
(Retinoblastoma), underlining that well-differentiated
pNETs and poorly differentiated pancreatic neuroendo-
crine carcinomas of small-cell and large-cell type are not
only clinically, but also genetically, different entities.8
Abbreviations used in this paper: ALT, alternative lengthening telomeres;
ATRX, ATR-X gene; CGH, comparative genomic hybridization; CI, confidence
interval; CIN, chromosomal instability; DAXX, death domain–associated
protein gene; Exp(B), odds ratio; FISH, fluorescence in situ hybridization; IHC,
immunohistochemistry; PML, promyelocytic leukemia; PNA, peptide nucleic
acid; pNET, pancreatic neuroendocrine tumor; TMA, tissue microarray.
© 2014 by the AGA Institute
0016-5085/$36.00
http://dx.doi.org/10.1053/j.gastro.2013.10.020
 454 Marinoni et al
Both DAXX and ATRX mutations in pNETs are associated
with alternative lengthening of telomere (ALT) activation.9
This phenotype is rarely observed in other types of carci-
nomas and never in pNETs with DAXX and ATRX
expression.10
ALT is by definition a telomerase-independent means of
telomere stabilization (reviewed by Bollmann11). Although
the mechanisms and the regulation of ALT are not
completely understood, homologous recombination of telo-
meric regions seems to be the major mechanism for telo-
mere lengthening in ALT activated cells. As a consequence of
telomeric recombination, a characteristic finding in ALT-
positive cells is the considerable heterogeneity of telomere
size with both very long and very short telomeres. The latter
can drive CIN (reviewed by O’Sullivan and Karlseder12).
Here, we show that DAXX or ATRX loss and ALT acti-
vation correlate with CIN in well-differentiated pNETs, and
that patients with DAXX- or ATRX-negative tumors have a
higher frequency of recurrence and a shorter survival time.
CLINICAL PANCREAS
Materials and Methods
Tissues, Patient Characteristics, and
Follow-Up Data
pNETs of 142 patients (collective I, University of Zurich)
were formalin-fixed and paraffin-embedded. A subset of
collected tissues additionally was snap-frozen. Clinical follow-
up data of 113 patients were obtained by contacting the pa-
tients’ general practitioners and Cancer Registry Zurich. The
tumors were reclassified into 3 groups according to the 2010
World Health Organization classification.13 In addition, 101
patients with follow-up data were selected from the Depart-
ment of Pathology of Bern (collective II). Tissue microarrays
(TMAs) comprising pNET tissues from both collectives were
used for the immunohistochemistry (IHC) and fluorescence in
situ hybridization (FISH) analysis. Only primary G1–G2 tumors
were considered in our analysis. The number of samples used
for the different analyses is reported in Supplementary Table 1.
The use of patient material was approved by the local ethics
committees (Bern: number 15-04-12; and Zurich: StV 40-2005).
DNA Extraction and DAXX, H3F3A, and
ATRX Sequencing
DNA was extracted from frozen tissues using the DNeasy
Blood and Tissue Kit (Qiagen, Hilden, Germany). DNA was
extracted only from samples with more than 80% tumor cell
content. Sanger sequencing for DAXX and H3F3A exon 1 mu-
tation screening was performed. All DAXX coding exons as well
as exon/intron boundaries were sequenced. Samples were cy-
cle sequenced using the Dye Terminator v1.1 cycle sequencing
kit (Applied Biosystems, Life Technologies, Grand Island, NY)
and read by the Applied Biosystems 3500 genetic analyzer
(Applied Biosystems). The primers used for the sequencing are
listed in Supplementary Table 2.
The ATRX gene was analyzed by semiconductive sequencing
using an Ion Torrent PGM (Life Technologies). Protein coding
exons were amplified by multiplex polymerase chain reaction
using 2 primer pools designed by the Ion AmpliSeq Designer
Gastroenterology Vol. 146, No. 2
(Life Technologies). Library construction, emulsion polymerase
chain reaction, and sequencing were performed according to
the manufacturer’s recommendations. The Torrent Suite 3.6.2
platform was used for sequence alignment with the hg19 hu-
man genome reference. Variant calling was performed with the
variant caller 3.6 software (Life Technologies, Grand Island,
NY). The average base coverage depth for most samples was
more than 2000 reads.
Immunohistochemistry for DAXX and ATRX
Four-micrometer sections were taken from the TMA and
stained with antibodies against DAXX (anti-DAXX, polyclonal
rabbit; Sigma, St. Gallen, Switzerland) and ATRX (anti-ATRX,
polyclonal rabbit; Sigma). The immunohistochemical staining for
all antigens was performed on an automated staining system
(Leica Bond III; Leica Biosystems, Nunningen, Switzerland).
Antigen retrieval for DAXX was performed by heating citrate
buffer at 100
for 30 minutes and for ATRX in 95
Tris buffer for
40 minutes. The primary antibody was incubated for 30 minutes
at a dilution of 1:40 for DAXX and a dilution of 1:400 for ATRX.
Visualization was performed using the avidin-biotin complex
method, which yielded a brown staining signal. For both DAXX
and ATRX scoring, only nuclear protein staining was considered
positive.7 To exclude false-negative samples, only samples with
positive nuclear staining of non-neoplastic cells and negative
tumor nuclei were scored as negative. Samples with both nega-
tive tumor nuclei and non-neoplastic stromal and endothelial
cells were scored as noninformative and excluded from further
analysis.
FISH
Four-micrometer TMA sections were cut. After deparaffi-
nization and rehydration, slides were boiled for 30 minutes in
citrate buffer, pH 7.2, and incubated for 30 minutes in 2 
standard saline citrate and 0.05% Tween 20. A peptide nucleic
acid (PNA) probe (telC-Alexa488; Panagene, Daejeon, Korea)
was diluted (1:10) in 70% formamide, 10 nmol/L Tris, pH 7.5.
One drop of PNA solution was spotted on hydrophobic gel bond
film and mounted on a glass slide. The samples were denatured
at 85
for 4 minutes and incubated for 2 hours at room tem-
perature in the dark. Slides then were washed in 60% form-
amide 10 nmol/L Tris, pH 7.5, for 5 minutes and 2  standard
saline citrate-Tween 20. Anti–promyelocytic leukemia (PML)
(antibody PG-M3; Santa Cruz, Heidelberg, Germany) 1:100 was
incubated for 1 hour at room temperature and the secondary
antibody (goat-anti-mouse Alexa568; Cell Signaling, Danvers,
MA) was diluted 1:500 and incubated for 1 hour at room
temperature in a dark chamber. One percent 40 ,6-diamidino-2-
phenylindole solution was incubated for 3 minutes at room
temperature. FISH was evaluated using an Olympus VS 110
Fluorescent Scanner (Olympus, Volkestwil, Switzerland).
Statistical Analysis
Univariate and multivariate Cox regression statistical ana-
lyses were performed with SPSS (version 21; IBM, Armonk,
NY), and Kaplan–Meier curves were plotted with GraphPad
Prism software (La Jolla, CA). The c2 test or the Fisher exact
test was used to calculate contingency tables. P values less than
.05 were considered statistically significant.
February 2014 DAXX and ATRX Loss in pNETs 455

Results found 9 new mutations in the coding region of DAXX and

DAXX and ATRX Protein Loss in pNETs
We performed IHC for DAXX and ATRX on a TMA with
142 pNETs (patient characteristics are shown in Table 1).
Only nuclear labeling for DAXX and ATRX were scored as
positive and only samples with positive internal control
were considered negative (Figure 1).7 Results were ob-
tained for 92 pNETs (24 tumors had no positive internal
control and no tumor tissue was evaluable in another 26
samples). We observed a loss of DAXX in 23 samples (25%)
and a loss of ATRX in 20 samples (18%). Altogether, 39
pNET samples showed loss of expression of at least 1 of the
2 proteins, with 4 cases showing a combined loss of both
DAXX and ATRX expression. Positive DAXX and ATRX nu-
clear staining was detected in 53 samples (57%). In 25
sporadic primary pNETs for which frozen tissue was avail-
able, we sequenced DAXX and H3F3A exon 1. ATRX
sequencing was performed from 16 tumor samples. We
3 mutations in the coding region of ATRX. No mutations
were found in H3F3A (Supplementary Table 3 and the
Supplementary Materials and Methods section). All the
mutations found in the DAXX and ATRX genes correlated
with loss of the corresponding protein expression in the
nucleus, except for the S403F mutation in the DAXX gene
and a H1722Y mutation in the ATRX gene, both of which
showed nuclear protein staining. In addition, in 4 cases with
loss of DAXX and in 2 cases with loss of ATRX protein
expression, no mutations in the corresponding gene coding
sequence were detected, suggesting either the presence of
DNA mutations in intronic and regulatory sequences or the
existence of alternative post-translational mechanisms
responsible for loss of DAXX and ATRX expression. DAXX
and ATRX nuclear labeling and ALT status for each
sequenced sample are reported in Supplementary Table 3.
ALT Activation in pNETs

CLINICAL PANCREAS
Table 1.Characteristics of the Two Patient Collectives To detect ALT activation in pNET tissues, we performed
PNA telomeric (Panagene) FISH together with PML immu-
Collective I Collective II nofluorescence on the TMA mentioned earlier. We detected
Total number of 142 101
30 ALT-positive samples and 33 ALT-negative samples.
patients Examples of ALT-positive and ALT-negative cases are re-
Average age, y 56 (141 patients; 59.59 (99 patients; ported in Figure 1. ALT-positive samples showed a mixture
no data for no data for of very bright large and very faint small telomeric signals, a
1 patient) 2 patients) hallmark of high heterogeneity in the size of telomeric se-
Sex quences. In 9 of the 30 ALT-positive samples (30%) we
Female 76 (54.2%) 46 (45.5%)
observed co-localization of PML and telomeric sequence at
Male 64 (45.8%) 53 (52.4%)
No data for No data for
the ALT, PML-associated nuclear bodies. In the remaining 21
2 patients 2 patients ALT-positive tumor tissues we observed heterogeneous
Grade telomeres without co-localization of PML in ALT, PML-
G1 108 (77.3%) 57 (56.6%) associated nuclear bodies owing to loss of PML expression
G2 32 (27.7%) 38 (37.6%) in the tumor cells. We observed loss of either DAXX or ATRX
No data for No data for expression in the majority (85%) of the ALT-positive cases,
2 patients 6 patients
whereas we found both DAXX and ATRX positivity in 70% of
Tumor stage
T1 46 (40.7%) 42 (41.5%) the ALT-negative samples (P ¼ .0003) (Table 2). Seven
T2 38 (33.6%) 16 (15.8%) pNETs showed loss of DAXX and ATRX expression without
T3–T4 29 (25.7%) 21 (20.7%) ALT activation; 6 of them were T1 tumors.
No data for No data for ALT-positive samples seemed to correlate histomorpho-
29 patients 22 patients logically with atypical cytology, defined as variation in nuclear
N stage size and chromatin density heterogeneity (Supplementary
N0 33 (61.1%) 44 (43.5%)
N1 21 (38.9%) 28 (27.7%)
Materials and Methods and Supplementary Figure 1).
No data for No data for
88 patients 29 patients
M stage DAXX or ATRX Loss and ALT Activation
M0 21 (55.2%) 60 (59.4%) Significantly Correlate With CIN
M1 17 (44.8%) 24 (23.7%)
To determine the relationship between ALT and CIN, we
No data for No data for
104 patients 17 patients
correlated the ALT phenotype with comparative genomic
Tumor specific 85.06 mo 75 mo (70 patients) hybridization (CGH) data of 67 pNETs previously
survival (mean) (79 patients) reported.6,14–19 pNETs with CIN were defined as showing a
No data for No data for total number of gains and losses of 8 or more in conven-
63 patients 19 patients tional CGH and of 20 or more in array CGH.17 CIN signifi-
Relapse free 103.44 mo 57 mo (64 patients) cantly correlated with DAXX or ATRX loss (P ¼ .0361). A
survival (mean) (85 patients)
significant correlation also was found between CIN and ALT
No data for No data for
57 patients 37 patients activation (P ¼ .0095). Statistical correlations (cross-tabu-
lation) between ALT, CIN, and DAXX and ATRX expression
are summarized in Table 2.
 456 Marinoni et al Gastroenterology Vol. 146, No. 2

Figure 1. Telomeric FISH,

DAXX, and ATRX immuno-
histochemistry of 3
different cases of pNETs.
Upper 2 rows: 2 ALT-
positive pNETs (cases 1
and 2), showing negative
DAXX (case 1) and negative
ATRX (case 2) nuclear la-
beling. In both cases stro-
mal cells show positivity for
DAXX and ATRX. Lower
row: example of an ALT-
negative case with DAXX
CLINICAL PANCREAS

and ATRX positive nuclear

staining (case 3). Only nu-
clear staining was consid-
ered for scoring and only
cases with an internal pos-
itive control were scored as
negative.

DAXX and ATRX Loss Associates In comparison with ALT-negative tumors, ALT-positive
With Prognosis tumors showed a significantly decreased relapse-free sur-
Collective I. We assessed whether DAXX or ATRX loss vival (log-rank, P ¼ .0015), as well as a reduced tumor-
would predict tumor recurrence as well as patient survival. specific survival (log-rank, P ¼ .018) (Figure 2B).
The Kaplan–Meier survival analysis was based on 61 pa-
tients for whom both IHC and clinical follow-up data were Prognostic Relevance of DAXX and ATRX
available. As illustrated in Figure 2A, nuclear loss of DAXX or
ATRX correlated significantly with both a shortened relapse-
Loss in an Independent Set of pNETs
free (log rank, P ¼ .0006) and tumor-specific survival (log-
Collective II. Because the analysis was performed on
a clinical case series we wanted to ensure that our findings
rank, P ¼ .0039).
were not caused by a bias in our collective. Thus, we
ALT activation correlated significantly with T stage
determined DAXX and ATRX expression in an independent
(P ¼ .0001). Of note, 28 of 29 (96%) ALT-positive tumors
additional consecutive case series including 101 pNET pa-
were larger than 2 cm whereas only 12 of 30 (47%) ALT-
tients undergoing surgery at the Inselspital Bern between
negative tumors reached such size (P ¼ .0001). TNM stag-
1987 and 2012. Follow-up data were available for 70 pa-
ing of ALT-positive and ALT-negative tumors is summarized
tients. Patient characteristics are summarized in Table 1.
in Table 3.
We detected a loss of DAXX in 8 samples (15%), and a loss
of ATRX in 15 samples (28%). In 2 samples both DAXX and
Table 2.Correlation Between ALT Activation, CIN, and DAXX ATRX were lost. In 33 samples (57%) DAXX and ATRX
and ATRX Nuclear Expression
expression were retained. In line with our observations,
P DAXX or ATRX loss predicted a decreased relapse-free
CIN- CINþ P value ALT þ ALT - value survival (log-rank, P ¼ .0003), but not a shortened tumor-
specific survival (log-rank, P ¼ .1778) (Figure 2C). Impor-
ALT tantly, because of the shorter follow-up time in this collection,
Positive 4 16 .0095 only 7 tumor-specific deaths were recorded in this collective.
Negative 13 7
DAXX/ATRX nuclear
staining
Positive 14 9 .0361 3 17 .0003
Combined and Multivariate Analysis
Negative 6 16 17 7 After confirmation of our observations in 2 independent
surgical pNET collectives, we pooled the 2 collectives to
perform a multivariate analysis to define the prognostic
February 2014 DAXX and ATRX Loss in pNETs 457

Figure 2. (A) Kaplan–Meier

curves of collective I de-
picting a significantly sho-
rter relapse-free survival
and a shorter tumor-
specific survival in DAXX-
or ATRX-negative pNETs
compared with DAXX- and
ATRX-positive pNETs. (B)
Kaplan–Meier curves of

CLINICAL PANCREAS
collective I depicting a
significantly shorter
relapse-free survival and a
shorter tumor-specific
survival in ALT-positive
pNETs compared with
ALT-negative pNETs. (C)
Kaplan–Meier curves of
collective II depicting a
significantly shorter
relapse-free survival in
DAXX- or ATRX-negative
pNETs compared with
DAXX- and ATRX-positive
pNETs.

Table 3.Association Between ALT Activation and value of DAXX or ATRX loss in comparison with the actual
Clinicopathologic Features gold standard prognostic markers used in daily clinical
practice. A univariate analysis of this pool showed multiple
Collective I ALT - ALT þ P value significant correlations between DAXX or ATRX and various
Sex clinicopathologic parameters (Table 4). Reduction of
Female 18 17 1 relapse-free survival was significant in both endocrinologi-
Male 15 12 cally defined subgroups of insulinoma as well as in
Tumor size, cm nonfunctioning tumors (data not shown).
<2 18 1 .0001 A Cox regression multivariate analysis including pa-
>2 12 28
rameters such as age, sex, stage, grade, and DAXX or ATRX
Grade
G1 29 19 .103
status was performed for tumor-specific survival, showing
G2 4 11 tumor grade as the only independent predictor (P < .001;
Tumor stage Odds ratio [Exp(B)], 8.941; 95% confidence interval [CI],
T1 18 1 .0001 2.798–28.573). The same analysis testing these parameters
T2 6 11 against patient relapse displayed 3 different parameters:
T3–T4 5 16 tumor stage (P < .011; Exp(B), 1.922; 95% CI, 1.161–3.184),
N stage
grade (P < .011; Exp(B), 2.949; 95% CI, 1.278–6.806), and
N0 11 7 .285a
N1 5 8 DAXX or ATRX loss (P < .028; Exp(B), 0.631; 95% CI,
M stage 0.418–0.952) (Supplementary Table 4).
M0 12 1 .0001
M1 1 9
Discussion
a
Probably artificial (see Supplementary material and We provide evidence that pNETs with loss of either
methods). DAXX or ATRX expression show ALT activation and CIN. No
 458 Marinoni et al Gastroenterology Vol. 146, No. 2

Table 4.Correlation Between DAXX and ATRX Expression malignant counterparts.6 Insulinomas with CIN have a poor
and Clinicopathologic Features outcome and CIN even has been proposed as a diagnostic
tool in those tumors.6
Collectives I þ II
In agreement with these data, DAXX or ATRX loss not
DAXX and ATRX þ DAXX or ATRX - P value only correlates with CIN, but also with tumor size and
metastasis. Our survival analysis showed that DAXX or
Sex ATRX loss is associated with a reduced relapse-free survival
Females 54 25 .010 and increased tumor-specific death.
Males 32 34 DAXX or ATRX loss proved to be an independent predictor
Tumor size, cm
<2 42 15 .004
for relapse, along with tumor stage and tumor grade, to affect
2 37 39 prognosis in a multivariate Cox regression analysis including
Grade well-established risk factors (tumor stage, tumor grade, age,
G1 67 35 .016 and sex). Notably, for tumor-specific survival only tumor
G2 19 25 grade remained significant in multivariate analysis. Grading
Tumor stage as used in the current American Joint Committee on Cancer/
T1 42 15 .001
Union internationale contre le cancer (AJCC/UICC) manual
T2 24 16
T3–T4 12 23
remains the prognostic factor with the highest risk ratio. Of
N stage note, DAXX or ATRX loss failed significance only marginally
N0 34 19 .100a (P < .051).
In line with the hypothesis that DAXX or ATRX loss leads
CLINICAL PANCREAS

N1 13 16
M stage
M0 59 29
M1 7 18
RFS mean, mo 85 49
TSS mean, mo 98 77
a
Probably artificial (see Supplementary material
methods).
specific genetic background has been associated previously
with CIN in pNETs. The significant association of loss of
DAXX or ATRX protein with ALT and CIN provides strong
evidence for a causal relationship between these factors.
Several arguments support this interpretation. ALT activa-
tion has been linked to CIN in several tumor types and in
glioblastoma with DAXX or ATRX loss.20–23 In vitro data
underline the possible link between ALT activation and CIN.
In a very recent study, Lovejoy et al24 showed that cell lines
with ALT activation and ATRX loss have a number of DNA
gains and losses comparable with the ones observed in
human cancer with CIN. Also, in vitro primary human and
murine ATRX-deficient myoblast cells show fragile telo-
meres and DNA damage after 96 hours in culture.25 In
primary cells and different cancer cell lines loss of DAXX and
ATRX sensitized cells to DNA damage and impaired DNA
repair,26,27 suggesting that DAXX and ATRX play an impor-
tant role in guarding genomic stability. However, it still has
to be clarified whether CIN is induced by ALT or whether
ALT is part of CIN; only mechanistic in vitro and in vivo
studies will be able to answer this question.
The correlation of DAXX or ATRX loss with CIN leads to
important clinical implications. Indeed, CIN is known to
characterize a clinically relevant subset of pNETs: in
nonfunctioning pNETs, large tumors are characterized by an
increased number of chromosomal aberrations, and metas-
tases contain more aberrations than their matched primary
tumors.15,16,28 In functioning pNETs, benign insulinomas
show significantly less aberrations as compared with their
to ALT and CIN and subsequently to an adverse outcome,
.001 we showed that ALT is associated significantly with a
decreased relapse-free and tumor-specific survival as well
.0001
.0019 as with tumor stage, size, and distant metastasis.
Interestingly, in small T1 tumors (<2 cm), only 14.2% of
DAXX- or ATRX-negative tumors showed ALT activation,
and whereas in larger (>2 cm) or metastasized tumors there
was an almost perfect correlation (of 17 pNETs negative for
DAXX or ATRX and ALT positive, only 1 was <2 cm). Heaphy
et al9 showed a perfect correlation: all their high-stage
pNETs with DAXX or ATRX loss showed ALT. These find-
ings together could suggest that DAXX or ATRX loss leads to
ALT activation only after some time. However, in vivo
models will be necessary to dissect the exact mechanisms.
We are aware that this study bears all the problems of a
retrospective study. Nevertheless, the availability of 2 in-
dependent and complementary pNET patient collectives
allows better exploitation of the strengths and reduces the
weaknesses of each one. Indeed, in collective I (including
patients undergoing surgery from 1973 to 2003) the
follow-up time is relatively long, allowing recording of a
sufficient number of death events, thus making this col-
lective powerful for survival analysis. In collective II
(including patients undergoing surgery from 1987 and
2012) the TNM data are more accurate owing to better
imaging tools and more sophisticated histologic work-up.
Our results seem to be in contradiction to the findings
reported by Jiao et al,7 who reported that 15 patients with
pNET carrying mutations in DAXX or ATRX genes had
appreciably longer survival than did 12 patients with wild-
type tumors. This discrepancy between our data and their
data could be owing to a different composition of the col-
lectives. Indeed, all 27 patients used in that study were
metastatic, as opposed to only 21 (18%) in our collectives.
Also, no stage T1 tumors were included in their collective
and 68% of them were stage T3 and stage T4, representing
an oncologic tertiary referral center situation and a palli-
ative setting. Our 2 study collectives were, instead,
composed of a more surgical than oncologic patient
February 2014
Figure 3. Representation of possible multistep tumor progression in pNETs. DAXX or ATRX mutations during uncontrolled cell
proliferation activate ALT and induce CIN. CIN induces genomic heterogeneity, allowing the selection of dominant subclones
of cells, and drives malignant tumor evolution and ultimately metastasis. Blue cells: cells carrying mutations in DAXX or ATRX,
ALT positive, and with CIN; green cells: metastatic clone.
selection, with 73% of the patients in stages T1 or T2 and
only 26% of them in stages T3 and T4. Of note, in a sub-
group analysis of 20 metastatic patients of our series,
DAXX- or ATRX-negative tumors also showed a trend
toward longer survival (Supplementary Materials and
Methods and Supplementary Figure 2). These data
together indicate that DAXX or ATRX loss might have a
different role in localized and distant tumor stages.
Several observations have indicated that DAXX or ATRX
loss could be a late event. In multiple endocrine neoplasia
1–associated pNETs, DAXX or ATRX loss was described only
in tumors larger than 3 cm and World Health Organization
grade 2.29 None of the microadenomas (<0.5 cm) analyzed
in the patients with multiple endocrine neoplasia 1 showed
loss of either DAXX or ATRX.29
If pNET progression goes from small benign lesions to
larger and malignant ones, the association of DAXX or ATRX
loss with large and metastatic pNETs suggests DAXX or
ATRX loss occurs as a late event.
DAXX or ATRX loss and ALT activation seem to be
secondary events that trigger progression from small
benign pNETs to large and malignant ones. Indeed, CIN
allows tumor cells to acquire a large spectrum of mutations
and chromosomal aberrations, conferring a selective ad-
vantage on subclones of cells, enabling their growth and
eventual dominance. A succession of such clonal expan-
sions would lead to multistep tumor progression (reviewed
by Hanahan and Weinberg30). In Figure 3 we model a
possible sequence of events leading to pNET malignant
transformation.
In conclusion, our study suggests DAXX or ATRX loss and
ALT as the causes of CIN in pNETs, and that they represent a
specific biological subtype of pNETs. These findings pave
the way to the understanding of the molecular mechanism
leading to CIN in pNETs and thereby to the identification of
new potential molecular targets.
Supplementary Material
Note: To access the supplementary material accompanying
this article, visit the online version of Gastroenterology at
www.gastrojournal.org, and at http://dx.doi.org/10.1053/
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DAXX and ATRX Loss in pNETs 459
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Received May 13, 2013. Accepted October 16, 2013.
Reprint requests
Address requests for reprints to: Aurel Perren, MD, University of Bern, Institute
of Pathology, Murtenstrasse 31, 3010 Bern, Switzerland. e-mail:
aurel.perren@pathology.unibe.ch; fax: (41) 31-632-49-95.
Acknowledgments
The authors thank Cornelia Schlup and Caroline Hammer for technical
assistance, Iria Gonzalez Vasconcellos for the fluorescence in situ
hybridization protocol and scientific support, Prof. P. Komminoth for helpful
discussions, and Inti Zlobec for careful reading of the manuscript. The
tissues were provided with support of the Tissue Bank Bern.
Conflicts of interest
The authors disclose no conflicts.
Funding
The study was supported by a Swiss National Foundation grant
(310030_144236 to A.P.); and supported in part by the Dutch Digestion
Foundation (E.-J.S.).
February 2014
Supplementary Materials
and Methods
DAXX and ATRX Sequencing
We sequenced 25 samples for DAXX and 16 samples for
ATRX (because DAXX and ATRX mutations are mutually
exclusive, we did not sequence ATRX genes in the samples
carrying mutations in the DAXX gene). As reported in
Supplementary Table 3, we detected 4 nonsense, 3 frame-
shift (deletions), and 2 missense mutations in the DAXX
gene. All the variants reported here, with the exception of
the 2 missense mutations, result in a premature stop codon
and in the translation of a truncated protein most likely
nonfunctional owing to a lack of the functional domain at
the C-terminal end.1 Interestingly, 1 of the 2 missense mu-
tations reported (p321 G>D) is located in a well-conserved
residue in the DAXX-H3.3 binding site and might impair the
interaction of the 2 proteins.2 Sequencing of the ATRX gene
gave rise to 1 insertion, resulting in a premature stop codon
and 2-point mutations in the helicase domain.
H3.3 mutations at codons 27 and 34 were examined in
25 samples; all 19 informative results were wild-type
sequence.
Survival Curves of M1 Patients
Survival curves of a subgroup of M1 patients from our
series are reported in Supplementary Figure 2. We could not
detect any significant difference in survival between DAXX/
ATRX-positive and -negative samples (P ¼ .616). However,
the significance for tumors with DAXX/ATRX loss for
adverse outcome is lost and DAXX/ATRX-negative tumors
even seem to have a trend for longer survival, in agreement
with what was reported by Jaio et al.3 It is important to
point out that we recorded only 3 death events in the DAXX/
ATRX-positive group and that the total number of M1 pa-
tients in our collective was rather small (only 20 patients:
15 patients were negative for DAXX or ATRX and 5 patients
were positive for both proteins).
DAXX/ATRX Loss and ALT Correlation
With N Stage
The N stage is the only parameter that does not correlate
either with DAXX/ATRX status or with ALT activation in the
combination of both our collectives. However, rather than
DAXX and ATRX Loss in pNETs 460.e1
being related to a biological reason, we believe this is
related to the historical work-up of our material. Indeed, as
reported in Supplementary Figure 3, the number of lymph
nodes analyzed increased with each year, indicating a
change in the surgical approach and/or pathologic analysis.
In agreement with these observations, if we include only
samples from 2000 to 2009 from collective II in the analysis,
which has the highest number of lymph nodes, DAXX/ATRX
loss correlates with N stage (P ¼ .0286) (Supplementary
Figure 3).
Morphology
We analyzed the morphologic characteristics of pNETs
with loss of DAXX/ATRX or ALT. By using a training set of
10 samples with known ALT and DAXX/ATRX status, we
defined the criteria for the scoring. We decided on nuclear
size variation, heterogeneity in chromatin density, and
presence of nodular fibrosis as potential markers. Twenty-
seven samples were included in the further blinded
analysis. A score of 0 (negative, agreement between 2 ob-
servers), 0.5 (no agreement between 2 observers), or 1
(obviously positive, agreement between 2 observers) was
assigned to each sample for each characteristic. No associ-
ation between any single variable and DAXX/ATRX loss or
ALT was found.
The combination of nuclear size variation and hetero-
geneity in chromatin density was able to predict ALT, but
not loss of DAXX/ATRX protein: samples with scores of 1.5
or greater considering nuclear size variation and heteroge-
neity in chromatin density correlated with ALT (P ¼ .0036).
Interestingly, both factors indicate cytologic atypia; such
atypical morphology seems to correlate with ALT. In
Supplementary Figure 1, an example of 1 ALT-positive and 1
ALT-negative pNET is depicted.
Supplementary References
1. Salomoni P, Khelifi AF. Daxx: death or survival protein?
Trends Cell Biol 2006;16:97–104.
2. Elsasser SJ, Huang H, Lewis PW, et al. DAXX envelops a
histone H3.3-H4 dimer for H3.3-specific recognition.
Nature 2012;491:560–565.
3. Jiao Y, Shi C, Edil BH, et al. DAXX/ATRX, MEN1, and
mTOR pathway genes are frequently altered in pancre-
atic neuroendocrine tumors. Science 2011;331:
1199–1203.
460.e2 Marinoni et al Gastroenterology Vol. 146, No. 2

Supplementary Figure 1. H&E staining of 2 pNETs cases. (A) ALT-negative pNET with homogenous nuclear size and chro-
matin density. (B) ALT-positive pNET with heterogenous nuclear size and chromatin density. This pNET shows cytologic
atypia.

Supplementary Figure 2. Kaplan–Meier curves of M1 pa-

tients from collectives IþII depicting a trend of DAXX/ATRX-
negative tumors toward a longer survival.
February 2014 DAXX and ATRX Loss in pNETs 460.e3

Supplementary Figure 3. Number of lymph nodes analyzed per patient over time. The number of lymph nodes increased with
time, either owing to changes in surgical technique or pathologic work-up, or both. Univariate analysis including only patients
from 2000 to 2009 from collective II showed correlation between DAXX/ATRX loss and N stage (P ¼ .0286).

Supplementary Table 1.Number of Samples Available for Each Analysis

Total samples: 243 IHC Mutation analysis Follow-up data Telomeric FISH CGH data

IHC 146 20 94 45 45
Mutation analysis 20 25 21 14 21
Follow-up data 94 21 149 42 48
Telo-FISH 45 14 42 63 40
CGH data 45 21 48 40 67
460.e4 Marinoni et al Gastroenterology Vol. 146, No. 2

Supplementary Table 2.Primer Sequences Used for DAXX

Exons and H3F3A Exon 1 Mutation
Analyses

Primer name Primer sequence Exon

Daxx 2aF 50 -CATTCTGAGGCCCCATCC-30 2

Daxx 2aR 50 -GCTTCTGCCCCAGGTGAG-30 2
Daxx 2bF 50 -GATGACGAAGATGAAGCA-30 2
Daxx 2bR 50 -AATCTTCCCCGCTAAAGCTC-30 2
Daxx 3aF 50 -CCCTTCCTTCTTCCCCTGA-30 3
Daxx 3aR 50 -CCCGAGACAGGACCCTAGA-30 3
Daxx 3bF 50 -CGGAGTTCTGCAACATCCTC-30 3
Daxx 3bR 50 -AGGTGTGTGGGAGGGTTATTC-30 3
Daxx 3cF 50 -CAATGAGCCCTCTGGGAATA-30 3
Daxx 3cR 50 -TCTGGGTCATCCAATTCTGAG-30 3
Daxx 3dF 50 -GGAAAAGGAGTTGGATCTCTCA-30 3
Daxx 3dR 50 -CTCAATGCGCCTGTTAACCT-30 3
Daxx 3eF 50 -ACCCGCTACCCAGAGGTT-30 3
Daxx 3eR 50 -CGTCGCTCCTGTAACCTGAT-30 3
Daxx 3fF 50 -AGGATGCCTTCCGAGATGT -30 3
Daxx 3fR 50 -TCCGCCTCATACCTGACATA-30 3
Daxx 4aF 50 -GGGCTGAGTGCTCTGACTTT-30 4
Daxx 4aR 50 -GGAGCCGAGCTCTTCTCTTT-30 4
Daxx 4bF 50 -CATGAGTCGGCTGGATGAG-30 4
Daxx 4bR 50 -AGTGCAGGGGAAGGACAAT-30 4
Daxx 5aF 50 -GGGCAAGGGATTCCTTGA-30 5
Daxx 5aR 50 -TGTTCCAGATCCTCCTCCTC-30 5
Daxx 5bF 50 -GACAGATGACGAAGACGATGAG-30 5
Daxx 5bR 50 -GAAATGACAGGTGAGGAGAATG-30 5
Daxx 6aF 50 -CCTCCTAGTGTCCTCTCAGCA-30 6
Daxx 6aR 50 -TGGTGACACTATGCGTCCTT-30 6
Daxx 6bF 50 -AGGGGAGCAGCAAAACAAA-30 6
Daxx 6bR 50 -CACAGAGGAAGGGGTATCCA-30 6
Daxx 6cF 50 -GAAGCTTTGCCCCTGGATAC-30 6
Daxx 6cR 50 -TGCTTCTTCTCCTTCCGAGA-30 6
Daxx 6dF 50 -CAACTGGGGAGATTCTGGTC-30 6
Daxx 6dR 50 -CCCAGTATTGCTCTCCGAAA-30 6
Daxx 7aF 50 -CCCGTACCCCTCCACATATT-30 7
Daxx 7aR 50 -GATGCAGAGGGAGCTGGTC-30 7
Daxx 7bF 50 -CCAGTTGCTGATTCCTCCA -30 7
Daxx 7bR 50 -AGCAGCTGAAACAGCCAAT-30 7
Daxx 8aF 50 -TCCTGTACTCCAGATCTCTTTGA-30 8
Daxx 8aR 50 -AGCTTAGTCCATCCCTTCCTC-30 8
H3F3AF 50 -GTGGTAAAGCACCCAGGAAG-30 1
H3F3AR 50 -CAAGAGAGACTTTGTCCCATTTTT-30 1
February 2014 DAXX and ATRX Loss in pNETs 460.e5

Supplementary Table 3.Mutation Analysis and Nuclear Expression of DAXX/ATRX and H3F3A

DAXX ATRX H3F3A DAXX IHC ATRX IHC ALT

c.1058 C>G; p.353 S>STOP codon Not sequenced WT X X X

c.1208 C>T p.403 S>F Not sequenced WT Pos Pos þ
c.963 G>A p.321 G>D Not sequenced X Neg Pos þ
c1063delC Not sequenced X Neg Pos X
c310G>T; p.104 Q>Stop Not sequenced WT Neg X þ
c.364-367 del AAGC; Not sequenced WT Neg Pos þ
c.293del CT Not sequenced WT Neg Pos -
c.1912 C>T; p.638 Q>stop Not sequenced WT Neg Neg þ
c1345 G>T p.452 E>STOP Not sequenced WT Neg Neg þ
WT WT X Neg Pos þ
WT WT WT Neg Neg X
WT WT X X X X
WT WT WT Neg X X
WT WT X Pos Pos þ
WT WT X Pos Pos X
WT WT WT X X X
WT WT WT X X þ
WT WT WT Neg X X
WT c.5520_5521insGAATGA, p.T1840fs*2 WT Pos Neg þ
WT WT WT Pos Pos X
WT WT WT Pos Pos -
WT c.5856A>G, p.D1719G WT Pos Neg þ
WT WT WT Pos Pos X
WT c.5864C>T, p.H1722Y WT Het Pos þ
WT WT WT Pos Neg X

NOTE. Results of DAXX and ATRX IHC and Telomeric FISH of the sequenced samples are shown. Pos, nuclear expression of
DAXX/ATRX; neg, no DAXX/ATRX expression in the nucleus; Het, heterogenous DAXX expression in the nucleus; þ, ALT
positive; -, ALT negative; X, no data; WT, wild type.

Supplementary Table 4.Multivariate Analysis of Collectives

I and II

P value Exp(B) 95.0% CI for Exp(B)

Relapse-free survival
T stage .011 1.922 1.161
Grade .011 2.949 1.278
DAXX/ATRX .028 0.631 0.418
Specific survival
Grade .001 8.941 2.798