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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: christofer.juhlin@ki.se

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

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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

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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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