ORIGINAL ARTICLE

A novel germline SDHB mutation in a gastrointestinal stromaltumor patient without bona fide features of the Carney–Stratakisdyad

Ricardo Celestino • Jorge Lima • Alexandra Faustino •

Valdemar Maximo • Antonio Gouveia •

Joao Vinagre • Paula Soares • Jose Manuel Lopes

Published online: 10 December 2011

� Springer Science+Business Media B.V. 2011

Abstract Gastrointestinal stromal tumors (GISTs) are the

most common mesenchyme neoplasms of the gastrointes-

tinal tract. Gain-of-function somatic mutations of the KIT

or PDGFRA genes represent the most prevalent molecular

alterations in GISTs. In Carney–Stratakis dyad, patients

portray germline mutations of the succinate dehydrogenase

subunits B (SDHB), C (SDHC) and D (SDHD) and develop

multifocal GISTs and multicentric paragangliomas (PGLs).

We herein report a novel germline SDHB mutation

(c.T282A—Ile44Asn) occurring in a 26 years-old patient

diagnosed with a spindle cell intermediate risk GIST that

did not present KIT/PDGFRA/BRAF gene mutations. Fur-

ther analyses revealed loss of the wild-type SDHB allele

and complete loss of SDHB expression in the tumor tissue.

After genetic screening of other family members, we

detected in the patient0s mother a SDHB mutation without

any clinical/laboratorial evidence of GIST or PGL. Alto-

gether, our findings (germline SDHB mutation with

absence of PGL in the index case and of GIST and/or PGL

in his mother) raise the possibility that this familiar setting

corresponds to an incomplete phenotype of the Carney–

Stratakis dyad.

Keywords Gastrointestinal stromal tumor � SDH �Carney–Stratakis dyad

Introduction

Gastrointestinal stromal tumors (GISTs) are the most

common mesenchyme neoplasms of the gastrointestinal

tract [1]. The tumor cells of GISTs are morphologically

and immunohistochemically similar to the interstitial

cells of Cajal (ICCs) [2, 3], consistent with a common

origin from ICCs stem cells or a ICCs phenotype

differentiation.

While GISTs may display unique clinical and pathologic

features, they usually share genetic alterations. Somatic

gain-of-function mutations of v-kit Hardy–Zuckerman 4

feline sarcoma viral oncogene homolog (KIT) or of plate-

let-derived growth factor receptor, alpha polypeptide

(PDGFRA) genes are the most prevalent molecular alter-

ations occurring in GISTs [2, 4, 5]. So far, KIT and

PDGFRA mutations were reported to be mutually exclu-

sive [4]. KIT mutations occur in 41–92% of GISTs [6–11],

and albeit they do not induce KIT overexpression, they

cause constitutional activation of this receptor tyrosine

kinase pathway [12]. KIT expression is reported in [95%

of GISTs, including tumors with wild-type KIT and most of

PDGFRA-mutated GISTs [13].

R. Celestino � J. Lima � A. Faustino � V. Maximo � J. Vinagre �P. Soares (&) � J. M. Lopes

Institute of Molecular Pathology and Immunology

of the University of Porto (IPATIMUP), Rua Dr. Roberto Frias

s/n, 4200-465 Porto, Portugal

e-mail: psoares@ipatimup.pt

R. Celestino � J. Vinagre

Institute of Biomedical Sciences Abel Salazar of the University

of Porto (ICBAS), 4099-003 Porto, Portugal

R. Celestino � J. Lima � V. Maximo � A. Gouveia � J. Vinagre �P. Soares � J. M. Lopes

Medical Faculty, University of Porto, 4200-319 Porto, Portugal

A. Gouveia

Department of Surgery, Hospital Sao Joao,

4200-319 Porto, Portugal

J. M. Lopes

Department of Pathology, Hospital Sao Joao,

4200-319 Porto, Portugal

123

Familial Cancer (2012) 11:189–194

DOI 10.1007/s10689-011-9499-x

GISTs occur as sporadically in 90% of the patients,

whereas in the remaining 10% they are familiar or part of

syndromes, including autosomal-dominant inheritance

pattern hereditary conditions. Germline mutations in KIT

and PDGFRA have been described in a few families pre-

senting multifocal GIST. GIST can also be part of the

tumor spectrum of the Carney triad (paragangliomas,

GISTs and pulmonary chondromas) [14], Carney–Stratakis

dyad (paragangliomas and GISTs) [15] and in the Neuro-

fibromatosis type I (neurofibromas, GIST and other lesions)

[16].

Concerning therapy, GISTs represents one of the most

successful examples of therapy with tyrosine kinase

inhibitors (TKI). The TKI Imatinib mesylate (STI571) is

a first line palliative therapy for advanced GIST, with

great efficiency in patients harboring KIT or PDGFRA

tumor somatic mutations [17]. Nevertheless, not all

GISTs harbor the same genetic status, and that a subset

of patients/tumors may not respond (primary resistance),

develop resistance (secondary resistance) or intolerance

to Imatinib mesylate therapy. Sunitinib was approved as

an alternative TKI, for the treatment of patients with

resistance or intolerance to Imatinib [18]. The patho-

genesis of resistance and/or intolerance reinforces the

concept that other molecular events, other than KIT

or PDGFRA mutations, may be implicated in GIST

tumorigenesis. Noteworthy, the BRAFV600E somatic

mutation was reported in 3–7% of KIT/PDGFRA wild-

type GISTs [19–21]. BRAFV600E mutations were reported

in KIT-mutated GISTs [22], as well as in one patient

with secondary Imatinib resistance without detectable

secondary mutations in KIT/PDGFRA [20]. Furthermore,

somatic mutations in the caspase-4 gene (CASP4) were

reported, with the CASP4L365V mutation being detected

in 18% of GISTs (wild-type and KIT/PDGFRA-mutated),

but without significant association with clinicopatholog-

ical features [23].

In the recently reported Carney–Stratakis dyad, where

patients develop multifocal GISTs and multicentric para-

gangliomas (PGL) [15, 24], molecular studies disclosed

the presence of germline mutations of the succinate

dehydrogenase subunits B (SDHB), C (SDHC) and D

(SDHD); GISTs occurring in these patients do not harbor

KIT or PDGFRA mutations [25]. Germline mutations of

the SDHB, SDHC and SDHD had been previously

reported in inherited PGL and pheochromocytoma (PCC)

[26–30], but not in familiar GISTs, until they were

described in patients with the Carney–Stratakis dyad [25,

31].

In this study we report a novel germline SDHB mutation

occurring in an otherwise unrecognized familial condition,

in which the index case presents a GIST without other

features of the Carney–Stratakis dyad.

Materials and methods

Case presentation

A 26 years-old man was admitted with a 3 days history of

melena, asthenia and no hematemesis. The patient had no

significant previous medical history, was on no regular

medication and did not report any familial history of gastric

malignancy. Clinical examination was unremarkable

except for paleness and a tachycardia of 110 bpm. Blood

tests showed an hemoglobin level of 5.6 g/dl with normal

urea and electrolytes, normal liver function and clotting

tests. An esophagogastroduodenoscopy revealed a polypoid

submucosal tumor in the lesser curvature of the distal

antrum, covered by normal mucosa with an ulcer on top of

the tumor. While he was stable, he received a total of 4

units of packed red blood cells. Endoscopic biopsy was

negative for tumoral cells and an endo-ultrasonography

(EUS) disclosed a hypoechoeic, homogeneous and well

circumscribed lesion, located in the 4th layer of gastric

wall. The staging work up (EUS; abdomino-pelvic con-

trast-enhanced computed tomography/CT; thoracic X-ray)

did not disclose any signs of distant disease.

A laparotomy on day 12 confirmed a large mass in the

pre-pyloric region, and a distal gastric resection with pri-

mary end-to-end gastro-duodenal anastomoses (Bilroth I)

was performed. The patient had an uneventful recovery

with no further bleeding.

The histopathology study depicted a spindle cell inter-

mediate risk GIST: 4.5 cm largest size, and a mitotic count

of 16/50 HPF. In the immunohistochemistry study, the

tumor cells displayed diffuse expression of CD117 and

CD34 in the absence of keratins, actin and S100 protein

expression. The resection margins were negative for tumor

cells (R0). The molecular analysis disclosed absence of

KIT/PDGFRA/BRAF gene mutations and the presence of a

SDHB germline mutation (see below). Then, a cervical-

thoracic-abdominal-pelvic CT scan (body CT) was per-

formed, and did not disclose any lesion suggestive of PGL.

Furthermore, 123I-metaiodobenzylguanidine (MIBG)

scintigraphy was also negative, as were the urine meta-

nephrines test (two measurements of 24 h urinary meta-

nephrine and normetanephrine). At last follow-up, 99

months after resection, the patient does not show any

evidence of recurrent disease.

Mutation screening

DNA was extracted from 10 lm paraffin-embedded sec-

tions of tumor tissue and corresponding adjacent no-tumor

tissue. Slides were microscopically examined, and tumor

areas were marked and carefully dissected under micro-

scopic observation. Dissected material was deparaffinized

190 R. Celestino et al.

123

in xylene, washed in ethanol, and rehydrated. DNA

extraction was performed using the Genomic DNA Puri-

fication Kit (Citomed, Lisbon, Portugal) according to the

manufacturer’s protocol. Germline DNA was extracted

from peripheral blood lymphocytes by the standard pro-

teinase K-SDS digestion and saline precipitation [32].

The presence of SDHB, SDHC and SDHD mutations

was evaluated by PCR and DNA sequencing was per-

formed as previously described by Lima et al. [33]. The

presence of mutations was validated by a second PCR

followed by direct sequencing.

Multiplex ligation-dependent probe amplification

(MLPA)

Germline and tumor DNA were analyzed for intragenic

deletions using multiplex-ligation dependent probe ampli-

fication (MLPA) assay (SALSA MLPA KIT, P226SDHD,

MRC-Holland b. v., Amsterdam, the Netherlands), accord-

ing to the manufacturer’s instructions. MLPA fragments

were discriminated in an ABI PRISM 310 Genetic Analyzer

(Applied Biosystem), and the resulting data was analyzed

using Coffalyser software (MRC Holland, Amsterdam, The

Netherlands). All MLPA results were reproduced at least

three times.

Immunohistochemistry for SDHA and SDHB proteins

Tissue sections with 2 lm thickness were deparaffinized,

rehydrated and pre-treated with 1xEpitope Retrieval Solu-

tion pH 9 (Tris/EDTA-based buffer containing surfactant)

(E7119; Leica Microsystems, Newcastle Upon Tyne,

United Kingdom) in a 98�C water bath during 20 min.

Immunohistochemical staining was performed with the

Envision G/2 System/AP (K5355; Dako, Glostrup, Den-

mark), according to the manufacturer’s instructions. The

tissue sections were incubated at 4�C overnight with SDHB

primary antibody (Complex II subunit 30 kDaIp mono-

clonal antibody, mouse, 1:600 dilution; MS203; Mito-

sciences, Eugene, Oregon, USA) and incubated 1 h with

SDHA primary antibody (Complex II subunit 70 kDaFp

monoclonal antibody, mouse, 1:1,250 dilution; MS204;

Mitosciences, Eugene, Ore, USA).

The APAP (alkaline phosphatase anti-phosphatase)

method was used for detection, and the samples were

developed with permanent red chromogen. The slides were

mounted using a water-miscible mounting medium, after

counterstaining with haematoxylin.

Negative and positive controls were used simultaneously to

ensure specificity and reliability of the staining process. Pre-

viously tested positive cases of oncocytic variant of papillary

thyroid carcinoma were used as positive controls. Omission

of the primary antibody was used as negative control.

Immunohistochemistry evaluation (positive/negative) was

performed independently by two observers (J.M.L. and V.M.).

Results

Our patient presented a KIT and PDGRFA wild-type GIST

without any known family history of GIST, PGL or PCC.

After sequencing the SDHB, SDHC and SDHD genes, we

identified a previously unreported germline point mutation

in the SDHB gene (c.T282A) in the genomic DNA of the

tumor, in non-tumor tissue as well as in the peripheral

blood of the patient. This mutation leads to the substitution

of an Isoleucine for an Asparagine at residue 44

(p.Ile44Asn) (Fig. 1), which is a highly conserved residue

across eukaryotes. Analysis of 174 chromosomes from

healthy blood donors, revealed that the SDHB p.Ile44Asn

mutation was absent in the samples tested.

The sequencing electropherogram disclosed that the

mutation was heterozygous in the blood and in non-tumor

tissue, whereas only the mutant allele was present in the

GIST tumor cells (Fig. 1), suggesting loss of heterozy-

gosity (LOH) of the normal allele in the tumor. This result

was further supported by MLPA analysis, which is con-

sistent with loss of the whole wild-type allele of SDHB

gene in tumor tissue (Fig. 2). MLPA analysis disclosed the

presence of both alleles in the non-tumor tissue and in the

peripheral blood of the patient.

Upon immunohistochemistry analysis, we observed

SDHA and SDHB protein expression in the patient’s nor-

mal gastric mucosa (Fig. 1); conversely, the SDHB-muta-

ted GIST showed complete loss of SDHB expression, while

retaining SDHA protein expression.

All patient’s first degree relatives (mother, 56 years-old;

father, 60 years-old; and the twin sisters, 31 years-old)

(Fig. 3) were tested for the SDHB p.Ile44Asn mutation in

DNA extracted from the peripheral blood. Only the patient’s

mother presented the p.Ile44Asn germline SDHB mutation,

but the subsequent clinical evaluation—a cervical-thoracic-

abdominal-pelvic CT scan and a 123I-metaiodobenzylgua-

nidine [MIBG] scintigraphy, as well as the 24 h urinary

metanephrines–showed no evidence of GIST and/or PGL, or

of any other lesion.

Discussion

In our study, we describe a novel germline SDHB mutation

occurring in a patient with an apparently sporadic GIST (no

known personal or family evidence of GIST, PGL or PCC)

without KIT or PDGFRA somatic mutations. Additionally,

the tumor tissue presented LOH of the wild-type SDHB

allele, consistent with the common two ‘‘hit’’ tumor-suppressor

A novel SDHB germline mutation in GIST 191

123

inactivation model. The SDHB p.Ile44Asn germline muta-

tion was also present in the patient’s mother, who, at last

follow-up is well and without evidence of disease. Our

results indicate that we are in face of a Carney–Stratakis dyad

with an incomplete penetrance as previously reported for

most SDHB germline mutations [33].

Carney–Stratakis dyad is a familial condition with an

autosomal dominant inheritance pattern with incomplete

penetrance, in which the development of GIST is associ-

ated with PGL [24, 25, 31]. Patients with Carney–Stratakis

dyad present germline mutations in SDHB, SDHC or

SDHD gene and do not harbor somatic or germline muta-

tions of KIT or PDGFR. The Carney triad is another con-

dition comprising GIST, PGL and pulmonary chondroma,

and without apparent familial history [14, 34]; the majority

of patients develop GIST and pulmonary chondromas [14]

Fig. 1 SDHA and SDHB immunoexpression and SDHB sequencing

results of the normal gastric mucosa (upper panel) and GIST tissue

(lower panel). Note the loss of SDHB immunoexpression in the GIST

tissue. Both electropherograms are evidencing the SDHB c.T282A

substitution in normal gastric mucosa and GIST, with loss of the wild-

type allele in the tumor. The mutated residue is indicated by an arrow.

Original magnification of images: 9200

Fig. 2 MLPA quantification of each SDHB exon in genomic DNA

extracted from the patient’s tumor tissue, normal gastric tissue and

peripheral blood; all SDHB exons show loss of DNA material in

tumor tissue when compared with normal tissue and peripheral blood.

Data was normalized using 5 genomic DNA control samples isolated

from normal human tissue. The bars represent the average of 3

experimental replicas

Fig. 3 Pedigree of the family with SDHB-mutated GIST

192 R. Celestino et al.

123

and so far no somatic or germline KIT/PDGFRA, SDHB,

SDHC and SDHD mutations have been detected in the

Carney triad [35, 36].

The case herein reported does not completely fit with a

Carney–Stratakis dyad, because there is neither evidence of

PGL in the patient nor in the mother who also carries the

mutation. It is possible that the absence of tumors in the

index patient mother may indicate an incomplete pene-

trance or a higher susceptibility of males to develop partial

(only GIST) features of the Carney–Stratakis dyad. Our

case fits with those recently described by Janeway et al.,

who identified germline mutations in SDHB and SDHC in

12% of patients with apparently sporadic GIST (no known

personal or family history of PGL) which also did not

disclosed KIT or PDGFRA somatic mutations [37]. Whe-

ther our case and those reported by Janeway et al. are cases

of the Carney–Stratakis dyad with reduced penetrance and/

or expressivity of the disease, or represent a new hereditary

GIST syndrome remains to be ascertained.

It was recently reported that the immunohistochemical

expression of SDHB strongly correlates with the genetic

status of SDH genes [38]: cases without germline SDH

mutations display SDHB expression, whereas cases har-

boring germline mutations in any SDH (B, C or D) gene are

consistently negative for SDHB expression [38, 39].

Accordingly, we observed that SDHB expression was

absent in the tumor cells, despite its expression in the

normal, non-tumor tissues of the patient. Ours results

reinforces the pathogenicity of the SDHB p.Ile44Asn

mutation and further supports the use of SDHB immuno-

expression as useful routine test for the identification of

GISTs with germline SDH mutations.

GISTs without KIT/PDGFRA somatic mutations repre-

sent a therapeutic challenge since it is known that they do

not respond well or may be resistant to treatment with

Imatinib [40, 41]. Considering the recent reports pointing

to SDH defects as an alternative molecular mechanism in

GIST tumorigenesis, therapies targeting SDH-induced

pathways might be considered in the medical decision of

Imatinib-resistant GISTs. Several promising drugs are

under trial evaluation, such as HIF1-a inhibitors and

derivatives of a-ketoglutarate or dichloroacetate (DCA).

In summary, we report a GIST patient without KIT/

PDGFR somatic mutations, who harbors a previously

unreported germline SDHB mutation. The absence of PGL

in the patient as well as the absence of GIST and/or PGL in

his mother (who carries the mutation) raises the possibility

that our case may represent an incomplete phenotype of the

Carney–Stratakis dyad or a distinct entity.

Acknowledgments This study was supported by Fundacao Calouste

Gulbenkian through a PhD grant to R.C.; by Fundacao para a Ciencia

e Tecnologia through the program Ciencia 2007 (V.M.) and 2008

(J.L.) and Novartis through a project grant. IPATIMUP is an Asso-

ciate Laboratory of the Portuguese Ministry of Science, Technology

and Higher Education that is partially supported by the FCT.

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