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Familial Cancer ISSN 1389-9600 Familial CancerDOI 10.1007/s10689-012-9520-z
Molecular characterization of parathyroidtumors from two patients with hereditarycolorectal cancer syndromes
Adam Andreasson, Luqman Sulaiman,Sónia do Vale, João Martin Martins,Florbela Ferreira, Gabriel Miltenberger-Miltenyi, Lucas Batista, et al.
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ORIGINAL ARTICLE
Molecular characterization of parathyroid tumors from twopatients with hereditary colorectal cancer syndromes
Adam Andreasson • Luqman Sulaiman • Sonia do Vale • Joao Martin Martins •
Florbela Ferreira • Gabriel Miltenberger-Miltenyi • Lucas Batista •
Felix Haglund • Erik Bjorck • Inga-Lena Nilsson • Anders Hoog •
Catharina Larsson • C. Christofer Juhlin
� Springer Science+Business Media B.V. 2012
Abstract The tumor suppressor adenomatous polyposis
coli (APC) has recently been implicated in parathyroid
development. We here report clinical, histopathological
and molecular investigations in parathyroid tumors arising
in two patients; one familial adenomatous polyposis (FAP)
syndrome patient carrying a constitutional APC mutation,
and one Lynch syndrome patient demonstrating a germline
MLH1 mutation as well as a non-classified, missense
alteration of the APC gene. We sequenced the entire APC
gene in tumor and constitutional DNA from both cases,
assessed the levels of APC promoter 1A and 1B methyla-
tion by bisulfite Pyrosequencing analysis and performed
immunohistochemistry for APC and parafibromin. In
addition, copy number analysis regarding the APC gene on
chromosome 5q21-22 was performed using qRT-PCR.
Histopathological workup confirmed both tumors as para-
thyroid adenomas without signs of malignancy or atypia.
No somatic mutations or copy number changes for the APC
gene were discovered in the tumors; however, in both
cases, the APC promoter 1A was hypermethylated while
the APC promoter 1B was unmethylated. APC promoter
1B-specific mRNA and total APC mRNA levels were
higher than in normal parathyroid samples. Immunohisto-
chemical analyses revealed strong APC protein immuno-
reactivity and positive parafibromin expression in both
parathyroid tumors. Absence of additional somatic APC
mutations and copy number changes in addition to the
positive APC immunoreactivity obtained suggest that the
tumors arose without biallelic inactivation of the APC
tumor suppressor gene. The finding of an unmethylated
APC promoter 1B and high APC 1B mRNA levels could
explain the maintained APC protein expression. More-
over, the findings of positive parafibromin and APC
immunoreactivity as well as a low MIB-1 proliferation
index and absence of histopathological features of
malignancy/atypical adenoma indicate that the parathy-
roid adenomas arising in these patients did not harbor
malignant potential.
Keywords APC � Parafibromin � Parathyroid �Carcinoma � Atypical adenoma
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10689-012-9520-z) contains supplementarymaterial, which is available to authorized users.
A. Andreasson � L. Sulaiman � F. Haglund � E. Bjorck �I.-L. Nilsson � C. Larsson � C. C. Juhlin (&)
Department of Molecular Medicine and Surgery,
Karolinska Institutet, Stockholm, Sweden
e-mail: [email protected]
A. Andreasson � L. Sulaiman � F. Haglund � E. Bjorck �C. Larsson � C. C. Juhlin
Center for Molecular Medicine CMM, Karolinska University
Hospital Solna, Stockholm, Sweden
S. do Vale � J. M. Martins � F. Ferreira
Endocrine Department, Santa Maria Hospital and Lisbon
Medical School, University of Lisbon, Lisbon, Portugal
G. Miltenberger-Miltenyi
Molecular Medicine Diagnosis Laboratory, Molecular Medicine
Institute, Lisbon Medical School, University of Lisbon, Lisbon,
Portugal
L. Batista
Department of Surgery, Santa Maria Hospital, Lisbon and
Lisbon Medical School, University of Lisbon, Lisbon, Portugal
A. Hoog � C. C. Juhlin
Department of Oncology-Pathology, Karolinska Institutet,
Karolinska University Hospital Solna, Stockholm, Sweden
123
Familial Cancer
DOI 10.1007/s10689-012-9520-z
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Introduction
Primary hyperparathyroidism (PHPT) is a common endo-
crine disorder, usually caused by a solitary adenoma and
more seldom by hyperplasia, an atypical adenoma or a
parathyroid carcinoma [1]. PHPT is characterized by
hypercalcemia and abnormal secretion of parathyroid hor-
mone (PTH), and the treatment is in most cases surgical
[2]. The MEN1 gene is mutated in multiple endocrine
neoplasia type 1 and subsets of sporadic parathyroid ade-
nomas. Familial and sporadic PHPT carcinoma and subsets
of atypical adenomas and cystic adenomas exhibit inacti-
vation of the HRPT2/CDC73 gene and its product parafi-
bromin is linked to the Wingless (Wnt) pathway [3, 4].
Recently, the adenomatous polyposis coli (APC) tumour
suppressor has been coupled to parathyroid tumorigenesis,
as absent APC expression has been demonstrated in the
majority of carcinomas and in subsets of atypical adenomas
that were investigated [5–7]. Furthermore, hypermethyla-
tion of the APC promoter 1A has been demonstrated in
parathyroid adenomas, although APC protein expression is
conserved in this entity possibly due to transcriptional
activity from the APC promoter 1B [8], as has been shown
for tumors in other tissues [9–12]. However, the involve-
ments of other inactivating mechanisms such as APC
promoter 1B methylation and posttranscriptional regulation
have so far not been explored. Constitutional mutations in
the APC gene cause the familial adenomatous polyposis
(FAP) syndrome, an autosomal dominant condition in
which the patients present with multiple colorectal ade-
nomatous polyps, eventually leading to colon cancer [13].
On the molecular level, APC has multiple functions and
APC mutations have e.g. been shown to pave the way for
dysfunctional degradation and subsequent activation of the
oncoprotein b-catenin, a central mediator of the Wnt sig-
naling cascade [14, 15]. A previous study has indicated a
possible connection between PHPT and colon cancer or
FAP. Nilsson et al. [16] found an overrepresentation of
colon cancer in PHPT patients in the Swedish population.
In one reported patient with a phenotype of FAP, MEN1,
and papillary thyroid carcinoma, the parathyroid tumor
displayed loss of heterozygosity for the wild-type APC
allele in addition to a constitutional APC mutation sug-
gesting a key role of APC in the tumor development [17].
In addition, a correlation between families with FAP and
parathyroid tumors has been previously reported [18].
Given the loss of APC in subsets of parathyroid tumors
and previous reports of PHPT in FAP patients, we aimed to
analyze additional parathyroid tumors with established
APC mutations. We here present two patients with con-
stitutional APC gene mutations who both developed a
parathyroid adenoma in addition to polyposis and colon
carcinoma. The parathyroid tumors were clinically
characterized and investigated for APC gene mutations,
copy number changes, promoter methylation as well as
APC and parafibromin immunohistochemistry.
Subjects and methods
Patient descriptions
Case 1 was a female Portuguese 78-year-old patient with
APC mutation-associated FAP for which she had subtotal
colectomy at age 48, followed by total colectomy a few years
later. Recently, the patient was referred to Santa Maria
Hospital, Lisbon, following detection of hypercalcemia at
routine blood analysis. She presented with fatigability,
weakness, depression and a densitometry verified trabecular
and cortical osteopenia in addition to a 30-years history of
urolithiasis. Her PTH levels ranged between 261 and
1861 ng/L (reference \70 ng/L), and her blood calcium
levels ranged between 9.7 and 13.3 mg/dL (reference
8.8–10.2 mg/dL). Although ultrasonic examination of the
neck failed to display parathyroid abnormalities, a subsequent
MRI scan revealed a left-side 28 9 10 mm heterogeneous
intra-thyroidal nodule, and the sestamibi scintigraphy indi-
cated an enlarged left inferior parathyroid gland located in the
superior mediastinum. Additional physical examinations,
routine blood analysis (including thyroid and pituitary func-
tion), ECG, ABPM (ambulatory blood pressure monitoring),
renal ultrasound and an adrenal CT scan showed no signifi-
cant abnormalities. However, since the patient displayed
raised basal catecholamines and a positive clonidine test, an
MIBG scintigraphy was performed to exclude the possibility
of a co-occurring pheochromocytoma. The scintigraphy was
negative, and pheochromocytoma was subsequently ruled
out. The patient was operated at age 78 and an enlarged
parathyroid gland measuring 21 9 11 9 9 mm was excised.
After surgery, serum calcium and PTH levels returned to
normal within days, while 1 month later calcium and PTH
levels were slightly increased. The APC mutation was also
observed in the patient’s daughter who also presented with a
FAP phenotype. Apart from the patient, no other family
members were known to have PHPT or other tumors sug-
gestive of multiple endocrine neoplasia type 1 or 2 (MEN1/2)
or the hyperparathyroidism-jaw tumor (HPT-JT) syndrome.
Case 2 was an 83-year old male patient who presented
with a history of polyps, colon cancer and Lynch syndrome.
The patient had an extensive family history of colon cancer
but was not aware of any family members suffering from
hyperparathyroidism. He had a history of polio in the
childhood and acquired a minor stroke in 1999. He had been
operated for colon cancer in 1969, 1976 and in 2002, in
addition to endoscopic removal of colorectal polyps on
some occasions; all of colon except for the rectum had been
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removed. The last operation had been associated with severe
postoperative complications with abdominal sepsis and
acute respiratory distress syndrome requiring tracheostomy
and mechanical ventilation. Recently, the patient was hos-
pitalized because of general weakness and was found to
have hypercalcemia with an ionised calcium of 1.61 mmol/
L (reference 1.14–1.32 mmol/L) and a PTH level of 200 ng/
L (reference \70 ng/L). Recapitulation of journal records
revealed an albumin corrected plasma calcium level of
2.93 mmol/L (reference 2.20–2.60 mmol/L) 6 years earlier
but this finding had not lead to any further investigation or
follow-up. Since preoperative ultrasound and sestamibi
scintigraphy were both inconclusive, probably due to a
simultaneous nodular goiter, a bilateral neck exploration
was performed. During surgery, an enlarged right upper
parathyroid gland was identified and removed. Two normal-
looking glands on the left side were identified. Scarring after
the earlier tracheostomy performance made the exploration
more difficult and the right inferior gland was not identified.
Postoperatively, the calcium levels normalized, the patient
recovered swiftly and left hospital on the second postoper-
ative day. Since long-standing primary hyperparathyroidism
was suspected, supplementation with calcium carbonate and
cholecalciferol was initiated promptly postoperatively. This
treatment needed to be maintained for more than 6 months
postoperatively to obtain normal plasma calcium levels and
to avoid symptoms of hypocalcemia.
Samples
Fresh frozen and paraffin-embedded parathyroid tumor
material and corresponding peripheral leukocytes were col-
lected at Hospital de Santa Maria, Lisbon, Portugal for Case
1 and at Karolinska University Hospital, Stockholm, Sweden
for Case 2. In addition, fresh-frozen samples of two atypical
adenomas (AtAd 44 and AtAd 46) previously shown to lack
APC immunoreactivity [6] and three reference parathyroid
samples (N1, N2 and N3) removed during thyroid surgery
without reimplantation were collected at Karolinska Uni-
versity Hospital, Stockholm Sweden. Samples were obtained
in accordance with local ethical guidelines and written
informed consent (Case 1) or oral informed consent docu-
mented in patient files and ethical approval at the Karolinska
University Hospital, Stockholm, Sweden (Case 2, AtAd 44
and 46 and the normal parathyroid tissues). Paraffin-
embedded material of parathyroid adenomas and anony-
mized colon cancer tissue were included as controls.
Histopathological assessment
All parathyroid tumor samples had been classified in rou-
tine histopathology at the respective hospitals following the
World Health Organization (WHO) criteria [1]. For this
study, the parathyroid tumors from Case 1 and Case 2 were
reviewed on hematoxylin-eosin stained sections by two
endocrine pathologists at the Karolinska University Hos-
pital, and MIB-1 proliferation index was determined by
routine Ki-67 immunohistochemistry.
DNA sequencing
The entire APC gene was sequenced in the tumor and blood
DNA from Case 1 and Case 2 as well as in tumor DNA
from AtAd 44 and AtAd 46. The methodology was carried
out essentially as described by Haglund et al. [19]. The
APC gene was amplified as 31 segments using PCR
primers described by Miyoshi et al. [20] or designed by the
clinical genetics department at Karolinska University
Hospital, Solna (Supplemental Table A). PCR products
were verified in 1.5% agarose gel stained with GelRed
agent and purified by Exosap-IT (USB Molecular Biology,
OH, USA). Sequencing was done at KIGENE Core Facility
of KI, utilizing an ABI 3730 capillary system (Applied
Biosystems, USA). All APC gene exons were successfully
sequenced with both forward and reverse primers, and
segments with suggested mutations were verified by repe-
ated sequencing. The mutation interpretation software
Alamut (Interactive Biosoftware, USA) was used for
evaluation of the APC mutation of case 2.
Sequencing of constitutional DNA from Case 1 has been
performed at the Molecular Medicine Diagnosis Labora-
tory at University of Lisbon for the APC, HRPT2 and RET
(exon 10, 11 and 16) genes. While HRPT2 and RET
sequencing revealed wild-type sequence only, a disease-
associated frameshift APC mutation was identified in the
patient and her daughter. Sequencing of constitutional
DNA from Case 2 has been performed at Department of
Clinical Genetics, Karolinska University Hospital which
revealed a disease-associated MLH1 gene mutation.
Bisulfite pyrosequencing
Sodium bisulfite modification of 500 ng total genomic
DNA was carried out following the manufacturer’s proto-
col (EpiTect Bisulfite kit, Qiagen AB, Sweden), and the
subsequent PCR and Pyrosequencing analysis to assess the
level of methylation were carried out as previously
described in detail by the authors in a preceding publication
[8]. The assays for APC promoter 1A and LINE-1 (Long
Interspersed Nuclear Element-1) are commercially avail-
able (PyroMark Assay Database, Qiagen). The APC pro-
moter 1B was identified based on a previously published
work [21] and the assay targeting APC promoter 1B was
designed using PyroMark Assay Design software version
2.0 (Qiagen) (Forward primer 50-GGAATAATGGA
TTAGTGTGTGTAGAAG, Reverse Primer biotinylated
Molecular characterization of parathyroid tumors
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50-CCCACAACCCCAAAACTAAAACCTATTATA, and
sequencing primer 50-AGATTAGGTTGTTTAGGTAG-
TAATG). To quantify the promoter methylation, the
average percentage of methylation at all the CpGs dinu-
cleotides analyzed was compared between the tumor and
the normal parathyroid samples analyzed. A difference of
10% in the average methylation level was regarded as a
significant change in the methylation status.
Quantitative RT–PCR (qRT-PCR)
DNA copy number assay
Copy number alterations at the APC gene locus in 5q21-22
were assessed using qRT-PCR (TaqMan Copy Number
Assay) as recommended by the manufacturer (Applied Bio-
systems, USA). Two assays targeting different parts of the
APC gene were selected: Hs02966112_cn overlapped the
intron 3-exon 4 junction and Hs03015312_cn targeted exon 6.
All samples were run in triplicates in a 96 well plate and in a
StepOnePlus realtime PCR machine (Applied Biosystems,
USA); and each experiment was repeated twice. After stan-
dardization against the RNase P gene used as internal refer-
ence, the tumor DNA was calibrated to commercially
available normal leukocyte DNA (Promega, USA). Data was
analyzed using the Sequence Detection Software SDS 2.2 and
the CopyCaller software V1.0 (Applied Biosystems, USA).
Gene expression assays
Parathyroid tumor RNA was available from Case 2 and sub-
sequently assessed for APC promoter 1B-specific as well as
total levels of APC mRNA (both promoter 1A and 1B) levels
using qRT-PCR. Reverse transcription reaction was per-
formed using High Capacity cDNA Reverse Transcription kit
(Applied Biosystems). Briefly, 100 ng RNA was used to
synthesize cDNA in a thermal cycler under the following
conditions: 25�C for 10 min, 37�C for 120 min and 85�C for
5 min. A total of 100 ng cDNA was used for subsequent
TaqMan gene expression qRT-PCR using the following
assays from Applied Biosystems: Hs01568269_m1 targeting
total APC mRNA, Hs01568282_m1 for the APC 1B-specific
transcript and RPLP0 (Hs04189669_g1) as an endogenous
control. Samples were run in triplicate in a 96 well-plate in a
StepOnePlus RT–PCR machine (Applied Biosystems) and
analyzed using SDS 2.4 software. The average Ct value was
normalized against the endogenous control and fold changes
calculated as DDCt value for each assay.
Immunohistochemical analyses
APC and parafibromin immunohistochemistry was carried
out as in our previously published reports [5, 6]. In short,
paraffin sections of the tumor samples were deparaffinized,
rehydrated, citrate treated, blocked in 1% BSA and incu-
bated overnight with the primary antibodies. The antibod-
ies used were rabbit monoclonal anti-APC (EP701Y;
Abcam, UK; 1:100) and mouse monoclonal anti-parafi-
bromin 2H1 (Santa Cruz Biotechnology, USA; 1:20).
Following incubation with biotinylated secondary anti-
bodies and the preformed avidin–biotin complex, samples
were incubated in DAB solution and counterstained using
haematoxylin. Parathyroid adenomas and omission of the
primary antibody served as positive and negative controls,
respectively, for the parafibromin antibody. For the APC
antibody, parathyroid adenomas were used as positive
controls, and colon cancer and omission of the primary
antibody functioned as negative controls. Furthermore, for
immunohistochemical analyses of microsatellite instability
markers in the parathyroid lesion of Case 2, antibodies
directed at MLH1, PMS2, MSH2 and MSH6 were
employed using established standardized protocols in the
clinical setting at the Department of Pathology at the
Karolinska University Hospital in Stockholm, Sweden.
Microsatellite instability (MSI) analysis
The parathyroid tumor DNA from Case 2 was analyzed
using microsatellite instability testing at the Department of
Clinical Genetics at the Karolinska University Hospital in
Stockholm, Sweden. The test employs five polymorphic
DNA markers targeting mononucleotide repeats (Bat 25,
26, NR 21, 24 and Mono 27).
Results
Detection of APC gene mutations
In Case 1, an APC gene mutation of exon 15 (c.3183-
3187delACAAA) was detected in the parathyroid tumor
DNA, which was identical to the mutation present in con-
stitutional DNA from the patient (Fig. 1). The mutation,
which involves loss of 5 nucleotides, was predicted to trun-
cate the full-length APC protein and give rise to a premature
stop codon 65 amino acids downstream of the deletion
(Gln1062X). The same mutation has previously been
reported as disease-associated in other FAP patients [20].
Furthermore, two known polymorphisms were observed:
Y486Y (rs2229992) and Y2401Y (rs2229994). In Case 2, an
APC gene sequence alteration in exon 15 (c.2534G[A) was
detected in both constitutional and tumor DNA of the patient
(Fig. 1). This alteration has not been previously described as
a mutation or as a SNP according to the databases NCBI,
Ensembl Genome Browser and UCSC Genome Bioinfor-
matics. The alteration is predicted to give a missense
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alteration at amino acid position 845 (Arg845His). As
determined from analysis of 11 species by Alamut this amino
acid is moderately conserved down to Xenopus tropicalis.
qRT-PCR analyses of the parathyroid tumors from Case 1
and Case 2 revealed no abnormalities in APC gene copies
(Table 1), in agreement with the heterozygous state of the
APC mutations detected.
In addition, we sequenced the entire APC gene in two
atypical parathyroid adenomas (sample AtAd 44 and 46)
previously shown to lack APC immunoreactivity [3].
Although no APC mutations could be detected, a number of
polymorphisms were found including: Y486Y (rs2229992),
R1678G (rs42427) and K1756S (rs866006) in sample 44;
and Y486Y (rs2229992), R545A (rs351771), R1493T
(rs41115) and R1960P (rs465899) in sample 46.
Promoter methylation density and APC gene expression
analyses
The parathyroid tumors from Case 1 and 2 were assessed
for DNA methylation at APC promoters 1A and 1B and
globally by analyzing LINE-1 repeats using quantitative
Fig. 1 Results from sequencing and immunohistochemistry in Case 1
and Case 2. At the top the APC mutations 3183-3187delACAAA
(Case 1), and 2534G[A (Case 2) are shown for the two parathyroid
adenomas. Below, positive APC and parafibromin immunoreactivity
is shown for both cases
Molecular characterization of parathyroid tumors
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bisulfite Pyrosequencing. All 10 assessed CpGs of APC
promoter 1A were hypermethylated giving mean methyl-
ation densities of 48 and 50% for Case 1 and 2, respec-
tively (Table 1). However, the 5 CpGs sites assessed for
APC promoter 1B only revealed low levels of methylation
with means of 3 and 6% respectively (Table 1). On the
global methylation level, tumor DNA from both cases
showed mean LINE-1 methylation levels of 74 and 70%
respectively, which were similar to the global methylation
levels revealed in reference parathyroid tissues (71%;
Table 1).
Gene expression analysis was performed in Case 2 using
primers specific to APC promoter 1B-derived mRNA and
total APC mRNA levels (from both promoters 1A and 1B).
This revealed higher expression (approximately 2.1-fold)
of APC 1B and total APC mRNA as compared to reference
parathyroid samples (data not shown).
Immunohistochemical analyses of APC
and parafibromin
Both cases demonstrated strong APC immunoreactivity in the
cytoplasm of all tumor cells using immunohistochemistry and
the EP701Y antibody (Fig. 1). Parafibromin expression was
evident using immunohistochemistry and the 2H1 monoclo-
nal antibody in which all tumor nuclei stained positive, also
with a weaker, concurrent cytoplasmic signal (Fig. 1). Posi-
tive controls consisting of unrelated parathyroid adenomas
were strongly positive for nuclear parafibromin and cyto-
plasmic APC immunoreactivity, respectively, while negative
controls displayed no or very little immunoreactivity.
Histopathological assessment
The parathyroid tumors of Case 1 and Case 2 were classified
as adenomas: Case 1 was a 21 mm cystic adenoma, and Case
2 a 7.1 gram partly cystic chief cell adenoma. At histopa-
thological re-evaluation the diagnosis of adenoma was
confirmed and features of atypical adenoma were excluded
such as marked nuclear atypia, trabecular growth pattern,
pleomorphism, fibrous bands, capsular engagement and
increased mitotic activity. Proliferation index was deter-
mined through routine Ki-67 immunohistochemistry
revealing less than 1% proliferation for both cases (Table 1).
MSI analyses
Immunohistochemical analysis of the parathyroid tumor
from Case 2 showed strong expression for MLH1 and
PMS2 (Supplemental figure 1) as well as for MSH2 and
MSH6 (data not shown). By contrast the surrounding
stroma and lymphocytes were negative or only weakly
stained as expected. Furthermore, testing for MSI in the
Case 2 parathyroid tumor did not reveal signs of MSI for
any of the five analyzed mononucleotide repeat markers.
Discussion
Lately, the APC tumor suppressor gene has been implicated
in parathyroid tumorigenesis, as the majority of carcinomas
and subsets of atypical adenomas display loss of APC
expression [5–7]. Although mutational analyses have failed
Table 1 Clinical and molecular
findings
Mean methylation in
parathyroid was determined
from three reference parathyroid
tissues (N1-3)
- Not investigated
Parameter Case 1 Case 2 Reference parathyroids
Clinical manifestations
Colon Colon cancer and polyps Colon cancer and polyps –
Parathyroid PHPT PHPT No
Parathyroid histopathology
Diagnosis Adenoma Adenoma Normal
MIB-1 index (Ki-67) \1% \1% –
Constitutional APC gene
Mutation 3183-3187delACAAA 2534G[A –
APC gene in PHPT adenoma
Mutation Gln1062X Arg845His –
Copy number assay No abnormality No abnormality –
Mean DNA methylation (range)
APC promoter 1A 48% (37–59%) 50% (36–61%) Mean 30% (23–36%)
APC promoter 1B 3% (2–5%) 6% (3–10%) Mean 7% (5–9%)
LINE-1 70% (67–75%) 74% (71–79%) Mean 70% (69–71%)
Immunohistochemistry
APC Positive Positive –
Parafibromin Positive Positive –
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to detect somatic APC gene mutations [6, 7], recent studies
imply that epigenetic silencing of the APC promoter 1A,
one of the gene’s two known promoter regions, is readily
observed in both parathyroid adenomas and carcinomas [7,
8]. As parathyroid malignant tumors readily display total
loss of APC expression for reasons which are still
unknown, there is emerging evidence that inactivation of
this tumor suppressor protein might influence malignant
behavior in parathyroid lesions.
In this study, we have analyzed APC and parafibromin
protein expression as well as the APC gene by DNA
sequencing, promoter methylation and copy number anal-
ysis in two parathyroid adenomas from patients endowed
with APC germline mutations. Both the histopathological
examination and the immunohistochemical markers (APC,
parafibromin and Ki-67) argued against malignancy in both
cases. The APC promoter 1A was found hypermethylated
(48 and 50% respectively) compared to preceding findings
in normal parathyroid tissue [7, 8] while the APC promoter
1B was found unmethylated. No somatic mutation of the
APC gene was found in the tumor tissue in any of the
patients. Copy number alterations were not revealed, i.e. no
losses or gains regarding the APC gene locus were detec-
ted. Consulting the current literature, our molecular find-
ings suggest that the parathyroid tumors investigated here
exhibit no malignant potential. Furthermore, the finding of
strong APC immunoreactivity argues against a biallelic
inactivation of the APC gene for these cases.
The retained APC expression in these two cases
endowed with one mutated allele and APC promoter 1A
hypermethylation suggests that APC transcription is pre-
served, which was also supported by TaqMan analyses of
Case 2 demonstrating high levels of APC 1B-specific
mRNA as compared to reference parathyroid tissues. This
is most likely due to unmethylated promoter 1B activity
and eventually also through insufficient amount of pro-
moter 1A methylation. Promoter 1B has previously been
shown to retain the ability to induce APC mRNA tran-
scription in various tumors, including parathyroid, despite
high methylation status of promoter 1A [8, 9]. The Py-
rosequencing analysis in this study revealed unmethylated
APC promoters 1B in both our cases, in accordance with
previous findings. It should, however, be stated that the
antibody used for APC immunohistochemistry targets the
N-terminal part of APC, and hence the detected immuno-
reactivity in the two parathyroid tumors in this study could
in theory stem from the truncated or missense mutated
APC variant respectively. More detailed protein analysis
could not be carried out due to the limitation of fresh
frozen material. However, previous studies using the APC
antibody EP701Y have found no or very little individual
discrepancies between wild type 311 kDa APC levels on
Western blot and APC immunohistochemistry results for
parathyroid tumors. Hence, the findings of positive APC
immunoreactivity with the EP701Y antibody may suggest
that the 311 kDa form of APC most likely is present in the
two parathyroid tumors investigated here [5, 6].
Case 2 is a parathyroid tumor derived from a patient
with Lynch syndrome presenting with a previous history of
colon cancer. Information from the patient’s medical charts
stated that the previously resected colon cancer stained
negative for MLH1/PMS2 and displayed microsatellite
instability using MSI analysis. We therefore sought to
determine whether or not the parathyroid adenoma from
the same patient also displayed evidence of MSI. The
results obtained suggest that the constitutional MLH1
mutation does not lead to an MSI phenotype in the para-
thyroid gland.
Given the association between parathyroid carcinoma/
atypical adenoma and loss of APC expression, the question
arise whether the APC mutated tumors studied here are
benign or harbor a malignant potential. The positive APC
and parafibromin immunoreactivity in the parathyroid
adenomas in addition to a low Ki-67 score and absence of
atypical histological findings would indicate that they did
not harbor malignant potential. Furthermore, the co-
occurrence of germline mutations of APC together with
APC promoter 1A hypermethylation is not to be considered
as a biallelic event in parathyroid tumors, as protein
expression is retained—probably via the APC promoter 1B.
However, as this report is based on two single observations
only, larger studies are needed to fully exclude a potential
correlation between malignant hyperparathyroidism and
germline APC mutations.
Acknowledgments The authors are truly obliged to the expert help
provided by Prof. Lars Grimelius of Karolinska University Hospital
Solna and Uppsala University Hospital for carefully reviewing the
parathyroid tumors by light microscopy. Furthermore, the authors are
indebted to Ms. Elisabet Anfalk for careful tissue management and
the Medical Genetics group for valuable discussions.
Conflict of interest The authors declare that they have no conflict
of interest.
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