Download Allelic Deletions on Chromosome 11q13 in Multiple Endocrine

ICANCERRESEARCH
57. 2238—2243.
June 1. 19971
Allelic Deletions on Chromosome 11q13 in Multiple Endocrine Neoplasia Type 1associated and Sporadic Gastrinomas and Pancreatic Endocrine Tumors
Larisa V. Debelenko, Zhengping Zhuang, Michael R. Emmert-Buck, Settara C. Chandrasekharappa,
Pachiappan Manickam, Siradanahalli C. Guru, Stephen J. Marx, Monica C. Skarulis, Allen M. Spiegel,
Francis S. Collins, Robert T. Jensen, Lance A. Liotta, and Irma A. Lubensky'
Laboratory of Pathology, National Cancer institute IL V. D., 1 1, M. R. E-B., L A. L, i. A. LI, National Centerfor Human Genome Research [S. C. C., P. M., S. C. G., F. S. C.],
and Branches of Metabolic Diseases (S. J. M., A. M. S.], Diabetes [M. C. S.), and Digestive Diseases [R. T. ii, National Institute of Diabetes and Digestive and Kidney Diseases,
N/H. Bethesda, Maryland 20892
ABSTRACT
Endocrine
tumors
(ETs)
of pancreas
and duodenum
occur
sporadically
and as a part of multiple endocrine neoplasia type 1 (MEN1). The MENJ
tumor suppressor gene has been localized to chromosome 11q13 by link
age analysis but has not yet isolated. Previous alleic deletion studies in
enteropancreatic ETs suggested MENJ gene involvement in tumorigenesis
of familial pancreatic ETs (nongastrinomas) and sporadic gastrinomas.
However, only a few MEN1-associated duodenal gastrinomas and spo
radic pancreatic
nongastrinomas
have been investigated.
We used tissue
microdissection to analyze 95 archival pancreatic and duodenal ETs and
metastases from 50 patients for loss of heterozygosity
(LOH) on 11q13
with 10 polymorphic markers spanning the area of the putative MENI
gene. Chromosome 11q13 LOH was detected In 23 of 27 (85%) MEN!associated
pancreatic
ETs (nongastrinomas),
14 of 34 (41%) MEN!-asso
ciated gastrinomas, 3 of 16 (19%) sporadic insulinomas, and 8 of 18(44%)
sporadic gastrinomas. Analysis of LOH on !!q!3 showed different dele
don patterns in ETs from different MEN! patients and in multiple tumors
from Individual MEN! patients. The present results suggest that the
MENJ gene plays a role in all four tumor types The lower rate of 1!q!3
LOH in MEN!-associated and sporadic gastrinomas and sporadic instill
nomas
as compared
to MEN!
nongastrinomas
may reflect alternative
genetic pathways for the development of these tumors or mechanisms of
the MENJ gene inactivation that do not involve large deletions. The
isolation of the MENJ gene is necessary to further define its role in
pathogenesis of pancreatic and duodenal ETs.
INTRODUCTION
MEN] is a tumor suppressor gene (9—11).MEN1 patients are hypoth
esized to inherit a mutation in one copy of the gene, and susceptible
cells in the target organs are transformed through the inactivation of
the wild-type copy of the gene, potentially occurring via point muta
tions, deletions, or gene methylation (6, 7, 10, 11). Sporadic parathy
roid and enteropancreatic ETs have also been described to exhibit
somatic LOH of chromosome 11 loci, including the MEN] region,
suggesting the role of the MEN] gene in the pathogenesis of such
tumors(12—19).
Allelic deletions on chromosome 11q13 have been reported in
63—100%of MEN1-associated parathyroid tumors and in 25—35%of
sporadic parathyroid tumors (1 1—13,20). However, previous studies
on 11q13 LOH in enteropancreatic ETs have been limited to a small
number of cases in each series (6, 13—19,21—24).Four MEN Iassociated gastrinomas have been reported in the literature to date, and
three tumors demonstrated retention of heterozygosity on 11q13 (17,
19, 24), whereas one gastrmnoma showed a small deletion at marker
PYGM (13). Thus, the role of the MEN] gene in enteropancreatic
endocrine
tumorigenesis
remains
controversial.
We used tissue microdissection to analyze 95 archival duodenal and
pancreatic ETs and metastases for LOH on 11q13. The goal was to
investigate the frequency of allelic loss at the MEN] gene locus in
tumongenesis of MEN1-associated and sporadic enteropancreatic
ETs. In addition, X-chromosome inactivation analysis of six synchro
nous primary duodenal microgastrinomas in one FMEN1 female
patient was performed to investigate clonality of MEN 1-associated
gastrinomas, and the results were correlated with the LOH data.
ETs2 of pancreas and duodenum may occur sporadically (I) or in
association with inherited syndromes such as MEN1 (2). Sporadic
duodenal
and pancreatic
ETs are usually
solitary,
whereas
MEN 1-
associated neoplasms are characteristically multiple in the involved
organ (3, 4). Insulinomas and nonfunctional ETs (nongastrmnomas)
occur exclusively in the pancreas, whereas the most common site for
both sporadic and familial gastrinomas is the duodenum (1—5).Insu
linomas usually follow a benign clinical course, whereas gastrinomas
have high malignant potential, with regional lymph node or liver
metastases
developing
in up to 90% of the cases.
The putative MENJ tumor suppressor gene has been linked to
chromosome
1 1q13 (6, 7). FMENI
is an autosomal
dominant
syn
drome in which the affected individuals develop multiple tumors in
the parathyroid glands (90—97%), pancreas (30—82%), duodenum
(25—60%),and anterior pituitary (35—60%;Refs. 2, 3, and 8). Loss of
the wild-type allele at the MEN] locus in tumors arising in affected
individuals is commonly observed, supporting the conclusion that
PATIENTSAND METHODS
Patient Population. Fifty patientswho underwentexploratorylaparotomy
for pancreatic and duodenal ETs at the NIH were included in the study.
Ninety-five formalin-fixed, paraffin-embedded primary pancreatic and duode
nal ETs and metastases were obtained from the file of the Laboratory of
Pathology, National Cancer Institute, NIH. Clinical and family histories were
reviewed in each case. Sixteen patients (9 males and 7 females; mean age, 45;
age range, 23—74
years) were diagnosed with MENI, and 34 patients (18 males
and 16 females; mean age, 42; age range, 15—67years) had sporadic ET.
Fourteen of 16 MEN! patients were categorized as having FMEN1 because in
addition to two typical endocrine neoplasms, they had at least two first-degree
relatives with MEN1-related endocrinopathies. The diagnosis of gastrinoma
(Zollinger-Ellison
nongastrinomas,
had sporadic
Received 1I/l 1/96; accepted 4/4/97.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I To
whom
requests
for
reprints
should
be
addressed,
at
Laboratory
of
Pathology,
National Cancer Institute, NIH, Building 10, Room 2N212. 9000 Rockville Pike,
Bethesda, MD 20892. Phone: (301) 496-0549; Fax: (301)480-9488.
2 The
abbreviations
used
are:
ET,
endocrine
tumor;
MENI,
multiple
endocrine
nan
plasia type I ; FMENI. familial MENI ; LOH, loss of heterozygosity.
syndrome),
insulinoma
(hyperinsulinemic
hypoglycemia),
or nonfunctional tumor was made on clinical grounds and confirmed by
pathological examination of the tumor. Seven MEN1 patients had pancreatic
I 1 MEN1
insulinomas
patients
had gastrinomas,
and gastrinomas,
respectively
and 16 and 18 patients
(Tables
1—4).Among
the 16 MEN! patients, 10 had gastrinomas only, 4 had pancreatic nongastri
nomas only, and 2 (patients 3 and 4) had both gastrinomas and nongastrinomas
available
for the study.
Nine MEN1
patients
had multiple
tumors
evaluated
(Tables 1 and 2).
Microdissection.
Tumor
and normal cells were selected
from routine
5-@tm-thickH&E-stained histological slides and microdissected under direct
light microscope visualization as described previously (Fig. 1; Refs. 20 and
2238
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GENETIC ALTERATIONS IN ENDOCRINE TUMORS
MEN!pancreaticendocrinetumors(nongastrinomas)3a4
Table 1LOHon!1q13in27
NFNFNFNFNFNFNFNFNFNFNFNFNFNFNFNF123456123478591011121314151617181920216
@4}@1516
In12 InInIn13
NFNFInc14
InNFb@4f@
.
I
I
D11S1256―
DllS956
DllS48O
I
D11S599
I
I
•
0
I
0
I
I
I
0
I
0
.
I
I
0
I
I
I
I
I
I
I
0
I
PYGM
I
I
I
0
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
—
I
I
I
I
I
I
I
I
I
• I
I
—
D11S4908
0
PPP1CA
I
I
I
0
I
0
D11S534
INT-2
I
I
• 0
I
I
•
I
—
a Patientno.
b Tumor type and tumor no. NF, nonfunctional; In, insulinoma; I, LOH; 0, retention of heterozygosity; —,not informative; blank, not done.
C Liver
metastasis.
d Chromosome
I 1q13
markers
are
listed
in
order
from
centromeric
(top)
to
telomeric
(bottom).
25). Normal duodenal epithelium, exocrine pancreas, or lymph node tissue was
used as a control.
DNA Extraction. Procuredcells were resuspendedin 30 @l
of solution
containing Tris-HCI (pH 8.0), 0.1 MEDTA (pH 8.0), 1% Tween 20, and 0.1
mg/mI proteinase K and incubated overnight at 37°C.Following thermal
inactivation ofproteinase K (95°C
for 5 mm), 1—1.5
pi ofthe DNA extract was
used for PCR analysis.
PCR Markers. Ten polymorphicDNA markerswere used in this study:
D11S1256
(26), D11S956,
D11S480
(27), D11S599
(28), D11S457
(D11S457,PYGM, and PPPJCA). Labeled amplified DNA was mixed with an
equal volume of fonnamide loadingdye (95% formamide,20 mxiEDTA, 0.05%
bromphenol
blue, and 0.05%
xylene cyanol).
The samples
were denatured
for 5
mm at 95°Cand resolved on a 6% polyacrylamide gel. Autoradiography was
performed with Kodak X-Omat film (Eastman Kodak, Rochester, NY).
The case was considered to be informative for a polymorphic marker on
Ilql3 if normal tissue DNA showed two alleles (heterozygosity). Complete or
near complete (90% decreased intensity) absence of an allele in tumor samples
was interpreted as LOH (Fig. 2). Each experiment was repeated two or three
(29),
PYGM (CA)(GA) (27), D1]S4908 (20), PPP]CA (30), D]1S534 (31), and
times,
INT2
Combined tumor and family study in a FMEN1 patient 8 with multiple
gastrmnomaswas performed with the informative marker D11S956 using nor
(27).
Labeling
[a-32P]dCTP.
of
PCR
product
was
achieved
by
incorporating
PCR was conducted in a total volume of 10 .d that contained
1—1.5
,.tl of DNA extract, 200 @M
each dNTP, 0.1—0.5 @M
each primer, 0.1
and the data were reproducible.
mal and tumor
DNA from the patient
and her brother
(Fig. 3).
X-Chromosome Inactivation Analysis. Six synchronous primary duode
The reactions were performedin a Perkin-ElmerCorp. thermal cycler as nal microgastrinomas from a female FMEN1 patient 2 (Tables 2 and 5) were
follows: denaturation at 94°Cfor 5 rain, followed by 35 cycles of annealing for studied for X-chromosome inactivation to assess clonality (Fig. 4). Extracted
45 5,extension at 72°Cfor 1 rain,anddenaturationat 94°Cfor 45 s. Annealing tumor and normal DNA (obtained from the same microdissection procedure as
temperatures for each set of primers were 62°C(D1]S599), 60°C(D11S599,), for LOH) was digested with HpaII (Life Technologies, Inc., Gaithersburg,
58°C(D]]S]256, D1]S599, D]]S480, D]]S4@YJ8,and 11(12), and 56°C MD) and amplified by PCR with primers to human androgen receptor
unit Taq DNA polymerase,
and standard PCR buffer (Perkin-Elmer
Corp.).
Table 2LOHon1
gastrinomasla
1q13 in 34 MEN!
4
j;;;
G G G G G G G G 0
In
lnIn
p
d
5
d
6
li d
7
d
d
8
d
9
d
d
Ii In In
•
I
•
1011
In
d
0
0
d
d
G G G G G GGGGGGGGGGGGGG000GGG
2d 3d 4d 5d 6d 7d 8ln 9In 10ln11In12d 13d
14DllSl256c 123 1516l7l8l920212223242526272829303l323334d
0
D11S956
D11S48O
0
—
0
0
D11S599
0
0
0
PYGM
D11S4908
0
I
I
0
0
0
0
0
• 0
• 0
0
•
0
0
S
0
0
•
•
0
0
0
0
0
0
——
0
—
•
•
0
•
0
0
0
—
S
•
I
0—
•
0
0
•
00
00
I0
00
0
0
•
0
0
•
0
•
0
00
00
00
00
00
00
00
S0
0.
0
0
0
•
0
I
•
•
0
0
PPPICA
0
0000
INT-20
00
——
I
• 0
• — 0
• • • 0
0
0
0
0
a Patientno.
b Tumor site, tumor type, and tumor no. ln, lymph node; d, duodenum; p. pancreas;li, liver, G, gastrinoma; @,
LOH; 0, retention of heterozygosity; —.not informative; blank, not
done.
C Chromosome
1 1q13
markers
are
listed
in
order
from
centromeric
(top)
to
telomeric
(bottom).
insulinomas35a363738394041424344454647484950InInInInInInInInInInInInInInInIn78910111213141516171819202122D11S1256―000D11S9560•0000•0—S0000—0D11S480—.——0————00
Table 3 LOH on 1iqi3 in 16 sporadic pancreatic
a Patient
no.,
tumor
type,
and
tumor
no.
In,
insulinoma;
•,LOH;
0, retention
of heterozygosity;
—, not
informative;
blank,
not done.
b Chromosome 11q13 markers are listed in order from centromeric (top) to telomeric (bottom).
2239
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GENETIC ALTERATIONS IN ENDOCRINE TUMORS
Table 4 LOH on !!q!3 in 18 sporadic gastrinomas
Patient no., tumor site, tumor type, and tumor no.
17
ln
18
In
19
In
20
ln
21
d
22
in
23
d
24
d
25
In
26
li
27
d
28
d
29
in
30
ln
31
d
32
p
33
li
34
li
G
G
G
G
G
G
0
G
G
G
G
G
G
G
G
G
G
G
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
0
0
0
—
I
I
0
—
0
0
0
I
0
—
0
0
—
—
o
—
—
—
—
—
0
I
—
0
—
—
I
D1IS599
—
—
I
—
0
—
—
—
I
—
—
0
I
—
—
DIIS457
PYGM
PPPICA
—
0
0
0
0
0
—
—
—
0
0
0
0
o
•
—
—
I
I
—
—
—
0
—
—
—
—
0
0
—
0
—
0
0
—
I
—
I
I
I
—
I
0
0
—
—
—
0
0
D1IS534
—
—
—
—
—
—
—
0
I
0
0
—
—
—
I
—
—
—
INT-2
0
0
—
0
0
I
0
0
—
0
0
0
I
0
I
0
0
a
I 1q13
DllS956a
DIIS48O
Chromosome
markers
are
listed
in order
from
centromeric
(top)
to telomeric
(bottom).
In,
lymph
node;
d, duodenum;
ii, liver;
p. pancreas;
—
G,
gastrinoma;
0
I,
LOH;
0,
retention of heterozygosity; —,not informative; blank, not done.
creatic MEN1-associated tumors included 6 insulinomas and 21 din
ically nonfunctional tumors (Table 1). Histological evaluation of
tumors revealed characteristic neuroendocrine features (Fig. 1) and
positive staining for chromogranin A (Boehringer Mannheim, Indian
apolis, IN) and/or synaptophysin (Zymed, San Diego, CA) by immu
nohistochemistry. The clinical diagnoses of gastrmnoma and insuli
A
NT
NT
D11S1256@
G13
G28
NT
NT
D11S956
D11S480
@
(HUMARA)
G36
G37
NT
NT
intensity
tissue
ui@
G39
G40
NT
NT
N T
(Fig. 4B).
Dl 1S457
NT
NT
N T
N T
N T
IPIT2
@-
1n7
Tumor Characteristics.Tissue microdissectionyieldedreliable
G47
•
.
DNA procurement from 95 tumors in 50 patients. Four groups of
tumors included: 27 pancreatic MEN1-associated ETs (nongastrmno
mas; sizes, 0.8—4.0 cm), 34 MEN1-associated gastrinomas (sizes,
0.5—8.0 cm), 16 sporadic insulinomas (sizes, 0.6—4.5 cm), and 18
NF4 NF1O
D11S534
G46
RESULTS
@
NT
•
following conditions published previously (32). The gastrinoma
in normal
NT
PPPICA
was considered to be monoclonal if PCR amplification from HpaII-digested
tumor DNA generated a single band as compared to two bands of equal
@
NT
D11S4908
D11S599
Fig. I. Multiple duodenal microgastrinomas (G8 and 09) in MEN1 patient 2 (H&E;
X20). A, before microdissection; B, after microdissection of tumor G9.
NT
PYGM
G41
@fl;
1n8
Fig. 2. Representativeresultsof llqI3 LOWretentionof heterozygositywith 10
polymorphicmarkersspanningthe area of the MEN! gene in MEN1-associatedand
sporadic ETs. G, gastrinoma; In, insulinoma; NF, nonfunctional tumor; N, normal tissue;
sporadic gastrinomas (sizes, 0.4—8.0 cm; Tables 1—4).The 27 pan-
T,tumor.Arrows,allelicdeletions.Numbers,tumornumbersin Tables1—4.
2240
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@
@
@+
@@1Mp4@@
GENETIC ALTERATIONS IN ENDOCRINE TUMORS
D11S956
@
I
èF@8
different LOll/retention patterns. For example, 4 of 14 gastrinomas in
patient 2 showed 11q13 LOH, whereas 8 tumors demonstrated reten
tion of heterozygosity with the markers tested (Table 2). Likewise,
patients 4 and 8 showed allelic deletions in two of three and three of
four tumors, respectively. Fig. 4A illustrates LOH results with marker
I
1
N G@G27G@PT
B
a
b@
D11S480
parathyroid tumor(Lane P7) from patient 8 (Pt 8)and normal peripheral blood DNA from
the patient'saffectedbrother(LaneB) wasamplifiedby PCRwithpolymorphicmarker
D11S956 and analyzed by denaturing gel electrophoresis. a, disease allele shared by
patient 8 and her affected brother; b, wild type allele. Arrow, all three of the patient's
gastrinomas (Lanes G26_28) and the parathyroid tumor (Lane P7) demonstrate a loss of
the wild-type allele b.
Table 5 Combined Iiq!3 WH and X-chronwsome inactivation results in six
(L)a
G, gastrinoma;
LOH
no
yes
no
no
yes
no
U, only
upper
allele
monoclonal (U)
monoclonal (L)
monoclonal (L)
polyclonal (U,L)
polyclonal (U.L)
monoclonal
detected
in tumor
as compared
to normal
tissue;
L, only lower allele detected in tumor as compared to normal tissue; U. L, both upper and
lower alleles detected in tumor.
immunoreactivity
MENI-associated
2.
X-ChromosomeInactivationin SynchronousMEN!-associated
three duodenal
gastrinomas
(Lanes
G4, G6, and G9) showed
a single clone (lower allele present), whereas gastrinoma G3 revealed
presence of the upper allele, indicative of a single but different clone.
Two tumors (Lanes G7 and G8) showed the presence of multiple
clones (both alleles present). Two alleles detected by X-chromosome
inactivation analysis in tumor G7 (with retention of heterozygosity by
LOH study) may represent a confluence of two separate clones,
whereas one of the two alleles in tumor G8 (allelic deletion by LOH
analysis) may represent a contamination with normal stromal cells
(Fig. 1; Refs. 20 and 25).
11q13 LOH in Sporadic ETs. Allelic deletions,spanningthe
with glucagon,
insulin,
or gastrin
(19%)
nongastrinomas
and
gastrinomas.
insulinomas
showed
deletions
in the area of the MEN]
gene.
in tumor
A
cells. Twenty-one insulinomas and 21 nonfunctional ETs were located
in the pancreas, and 1 ET (InS) represented insulinoma metastasis to
the liver (Tables 1 and 3). Among 52 gastrinomas, 26 were located in
the duodenum, 2 were in the pancreas, 19 were in the peripancreatic
lymph nodes, and 5 were liver metastases (Tables 2 and 4). The tumor
was considered to be malignant if regional lymph node or distant
metastases were documented. All 52 gastrinomas and 2 insulinomas
(1n3 and InS) were classified as malignant, and 21 nonfunctional
tumors and 19 insulinomas were benign. The study included 19
gastrinomas (G2—17and G30—32)and 6 nongastrinomas (1n2—5and
NF7—8)from 5 MEN1 patients (patients 2, 3, 9, 12, and 13) analyzed
in part in a previous report (20).
11q13 LOll In MEN1-associated ETs. Allelic deletions, span
ning the MEN] gene region on chromosome llql3, were detected in
both
in patient
MEN] gene region on chromosome 11q13, were detected in both
sporadic insulinomas and gastrinomas. The LOH results in 16 spo
radic insu!inomas are summarized in Table 3 and Fig. 2. Three of 16
noma were confirmed by positive immunostain for gastrmn (DAKO
Corp., Carpinteria, CA) and insulin (BioGenex, San Ramon, CA),
respectively. Most nonfunctional ETs stained positively with pancre
atic polypeptide immunostain (DAKO Corp.) and showed variable
scattered
microgastrmnomas
Gastrinomas.
DNA from the six synchronous microgastrinomas in a
female FMEN1 patient 2 procured during the same microdissection
procedure was analyzed for clonality using X-chromosome inactiva
tion assay (32). Separate tumors dissected from the patient's duode
num revealed different clonality patterns (Fig. 4B and Table 5). For
example,
synchronous duodenal gastrinomas in female patient 2 with MEN!
G4
06
G7
G8
09
duodenal
four gastrmnomas (Lanes G3, G6, G7, and G9) showed retention of
heterozygosity. All synchronous tumors from each patient with LOH
showed loss of the same allele (Fig. 4A).
Fig. 3. Combined pedigree and tumor deletion data in FMENI patient 8. Extracted
DNA from normal lymph node (Lane N), three gastrinomas (Lanes G26_28), and one
Tumorno.
ClonalityG3°
in six synchronous
@4—
Two
ofthe
six
tumors
(Lanes
G4*
and
G8*)
showed
allelic
loss,
and
The
N
N
G3G4G5G6G7G8G9
B
N N G3 G4 G5 G6 G7 G8 G9
LOH
results in 27 MEN1-associated pancreatic nongastrinomas from seven
patients are summarized in Table 1, and representative examples are
shown in Fig. 2. For each tumor studied, at least two polymorphic
markers were informative. Twenty-three of 27 (85%) nongastrmnomas
in six patients showed deletions in the area of the MEN] gene. LOH
was demonstrated in 4 of 6 insulinomas and in 19 of 21 nonfunctional
pancreatic ET.
Fourteen of 34 (41%) gastrmnomas in 6 MEN! patients studied with
8 polymorphic markers showed deletions in the area of the MEN]
gene (Table 2 and Fig. 2). LOH was detected in 9 of 20 duodenal
gastrinomas, 1 of 1 pancreatic gastrinoma, 3 of 11 lymph nodes, and
1 of 2 liver metastases, regardless of size of the tumor.
Multiple synchronous tumors in two MEN! patients with pancre
atic nongastrmnomas (Table 1, patients 3 and 13) and in three MEN1
patients with gastrmnomas (Tables 2 and 5, patients 2, 4, and 8) showed
Fig. 4. Combined I1q13 LOH and X-chromosome inactivation analysis in six syn
chronous duodenal microgastrinomas in female patient 2 with FMENI . N. normal duo
denal epithelium; G, gastrinoma. Nunthers, tumor numbers in Table 2. Arrowheads,
alleles. A, LOH results with marker Dl 1S480. Compared to normal tissue N, the lower
allele is lost in tumors G4° and G8° and retained in tumors G3, G6—7,and G9.
Synchronous tumors show loss of the same allele. B. X-chromosome inactivation results.
Tumor and normal DNA digested with HpaII was amplified by PCR with primers to
human androgen receptor (HUMARA). Three duodenal gastrinomas (G4. Gd. and G9)
show a single clone (lower), whereas G3 reveals presence of the upper allele, indicative
of a single but different clone. Two tumors (G7 and G8) show the presence of several
clones (both alleles present).
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GENETIC ALTERATIONS IN ENDOCRINE TUMORS
gastrinomas)AuthorTable 6 Literature data on !!q!3 LOll in MEN! and sporadic enteropancreatic ETs (nongastrinomas and
NongastrinomaGastrinomaLarsson
(Ref.)MENISporadicYear
2/2'@Bystrom
ci al. (6)1988
1/1Radfordet al.(21)1990
2/2Teh et aL (22)1990
1/1Patel
ci aL (14)1990
1/3Bale
et aL ( I5)1990
0/20/6Sawicki
et al. ( I7)199
1Ding et al. (18)19924/1
1/2Weber
el al. (16)1992
Nongastrinoma
Gastrinoma
1/1
1
3/4
0/1
2/2Eubanks
et al. (23)1994
2/58/22Beckers
el al. ( 19)1994
0/1Iwasakiel al. (24)1994
0/1
1/1Total1(13)1995
312/39Debelenko
1/12
1/4
et al.
23/27
14/34
3/16
(85%)
(41%)
(19%)
the number1997
All tumors in this group were located in the pancreas and were
clinically benign, and no correlation between the size of the tumor and
11q13 LOH was noted.
LOH results in 18 sporadic gastrinomas are summarized in Table 4
and Fig. 2. Eight of 18 (44%) ETs in sporadic gastrinoma group
showed deletions in the area of the MEN] gene. All tumors were
malignant. LOH was detected in three of six duodenal and five of
eight lymph node gastrinomas. Retention of heterozygosity on mul
tiple markers was seen in one pancreatic gastrinoma and three gastri
noma liver metastases. No relationship between the size or site of
gastrmnoma and the presence of deletions was observed.
Combined Tumor and Family Study in a FMEN1 Patient with
Gastrinomas.
To assess whether duodenal gastrinomas share the
same developmental mechanism with parathyroid tumors in MEN1
patients, DNA from normal lymph node tissue (Lane N), three duo
denal gastrinomas (Lanes G2628), and one parathyroid tumor (Lane
PT) in patient
8 and normal
peripheral
blood DNA
(Lane B) from the
affected brother was amplified with marker D]]S956 (Fig. 3). Patient
8 was heterozygous for D]]S956, and her normal DNA (Lane N)
contained two alleles, a and b. Her brother was homozygous for allele
a at D]]S956 (Lane B). Patient 8 and her affected brother sharedallele
a, and allele a was retained in all three of the patient's gastrinomas
(Lanes G26_28) and parathyroid tumor (Lane PT), whereas allele b
(derived from the unaffected parent) was lost in all four tumors. The
results are consistent with a tumor suppressor gene function of the
gene.
DISCUSSION
The MEN] gene has recently been localized between markers
D]]S]883
(44%)
of tumors studied showing I 1q13 LOH; denominator, the total number of tumors studied in each series.8/18
a Numerator,
MEN]
5/1
and D]]S449
on chromosome
1 1q13 by recombination
studies (33, 34). Tumor allelic deletion mapping data placed the gene
between markers PYGM and D]]S97 (13, 20, 21 , 23). The present
study represents the largest series of MEN1-associated and sporadic
enteropancreatic ETs analyzed with 10 markers in the area of the
putative MEN] gene to date (Table 6). On the basis of the minimal
region of overlapping deletions on I 1q13 in MEN1-associated tumors,
the MEN] gene boundaries are placed between markers D]]S480
(patient 5, 021) and INT2 (patient 2, G8 and G1 1, and patient 7,
G23—25; Table
2). The data in the present
study are consistent
with
previously reported boundaries in MEN1 tumors. The LOH analysis
of sporadic gastrinomas suggests that the MEN] gene may be distal to
PYGM (patient 21, G39, Table 4).
The present results suggest the MEN] gene is involved in tumori
genesis of MEN 1-associated pancreatic nongastrinomas and gastrino
mas, sporadic gastrinomas, and some sporadic insulinomas. All four
groups of tumors showed large deletions in chromosome 11q13 re
gion. Interestingly, varying LOH rates were observed among the four
groups of ETs. The lower incidence of 11q13 LOH in MEN 1-asso
ciated gastrinomas and metastases (41%), sporadic gastrmnomas
(44%), and sporadic insulinomas (19%) compared to a high LOH rate
in MEN1-associated pancreatic nongastrmnomas (85%) may indicate
that gastrinomas and some sporadic insulinomas could arise due to
inactivation of the wild-type allele via point mutations or small
deletions rather than via a loss of large segment of chromosome
11q13. Identification of the MEN] gene will allow testing of this
possibility. Alternatively, because a 19% (3 of 16) LOH rate detected
in benign sporadic insulinomas in the present study is low and similar
to a 17% (1 of 6) rate reported in this tumor type to date (15, 17, 19),
it is conceivable that the MEN] gene is inactivated in only a minority
of sporadic insulinomas. Because gastrinomas are the only common
MEN1-associated and sporadic enteropancreatic ETs with malignant
potential, in addition to initial inactivation of the MEN] gene, further
genetic alterations are probably necessary for their development and
progression. Therefore, gastrinomas may offer a unique opportunity to
study a chain of genetic events necessary for development of malig
nant neoplasm in a setting of familial syndrome.
The role of other tumor suppressor genes in enteropancreatic en
docrine tumorigenesis presently remains uncertain. Analysis of spo
radic pancreatic ETs for VHL gene (3p25.5) mutations by Chung et a!.
(35) revealed no evidence of the role of the gene in their development.
Furthermore, the screening of 27 MEN!-associated
and sporadic
pancreatic ETs (insulinomas and gastrinomas) with polymorphic
markers in the areas of known tumor suppressor genes on chromo
somes ip, 8p, and 3p in our laboratory did not demonstrate significant
rate of LOH (data not shown). Further studies are necessary to provide
a better understanding of enteropancreatic endocrine tumorigenesis.
In this study, multiple synchronous nongastrinomas and gastrmno
mas were analyzed in nine MEN! patients. In five of the patients,
varying deletion patterns were documented in enteropancreatic ETs
arising simultaneously regardless of tumor size, type, and anatomical
location. Correlation of the LOH and X-chromosome inactivation data
in six duodenal microgastrinomas from a FMEN1 female patient 2
demonstrates that each individual gastrinoma arises as an independent
event (Fig. 4 and Table 5). Moreover, each clonal tumor may show
either loss or retention of heterozygosity with a given marker tested
(Fig. 4A). The data supports the hypothesis that the mechanism of the
MEN] gene inactivation in gastrinomas may involve small deletions
or mutations. These results stress the importance of analysis of mul
2242
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GENETIC ALTERATIONS IN ENDOCRINE TUMORS
tiple tumors from each target organ in the study of pathogenesis of
hereditarycancer syndromesand in allelic deletion mappingof the
suppressor genes such as MEN] (20, 36). In view of the lower rate of
large deletions, MEN1-associated gastrinomas may represent a useful
resource for deletion mapping of the MEN] gene if the wild-type
allele is inactivated by small deletions. Furthermore, because the rate
and pattern of al!eic deletions on 1!q!3 is similar in sporadic gastri
nomas and MEN1-associated gastrinomas, the LOH analysis in the
former group may prove helpful to further define a minimal overlap
ping deletion interval in mapping of the MEN] gene.
18. Sawicki, M. P., Wan, Y-J. Y., Jonson, C. L., Berenson, J., Gatti, R., and Passaro, E.
Loss of heterozygosity on chromosome I I in sporadic gastrinomas. Hum. Genet., 89:
445—449,
1992.
19. Eubanks, P. J., Sawicki, M. P., Samara, G. J., Gatti, R., Nakamura, Y., Tsao, D.,
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Allelic Deletions on Chromosome 11q13 in Multiple Endocrine
Neoplasia Type 1-associated and Sporadic Gastrinomas and
Pancreatic Endocrine Tumors
Larisa V. Debelenko, Zhengping Zhuang, Michael R. Emmert-Buck, et al.
Cancer Res 1997;57:2238-2243.
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