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 1. nature
 2. modern pathology
 3. original article
 4. article

SDHB/SDHA immunohistochemistry in pheochromocytomas and paragangliomas: a
multicenter interobserver variation analysis using virtual microscopy: a
Multinational Study of the European Network for the Study of Adrenal Tumors
(ENS@T)
Download PDF
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 * Published: 27 February 2015


SDHB/SDHA IMMUNOHISTOCHEMISTRY IN PHEOCHROMOCYTOMAS AND PARAGANGLIOMAS: A
MULTICENTER INTEROBSERVER VARIATION ANALYSIS USING VIRTUAL MICROSCOPY: A
MULTINATIONAL STUDY OF THE EUROPEAN NETWORK FOR THE STUDY OF ADRENAL TUMORS
(ENS@T)

 * Thomas G Papathomas1,2,
 * Lindsey Oudijk2,
 * Alexandre Persu3,
 * Anthony J Gill4,5,
 * Francien van Nederveen6,
 * Arthur S Tischler7,
 * Frédérique Tissier8,9,
 * Marco Volante10,
 * Xavier Matias-Guiu11,
 * Marcel Smid12,
 * Judith Favier13,14,
 * Elena Rapizzi15,
 * Rosella Libe8,
 * Maria Currás-Freixes16,
 * Selda Aydin17,
 * Thanh Huynh18,
 * Urs Lichtenauer19,
 * Anouk van Berkel20,
 * Letizia Canu15,
 * Rita Domingues21,
 * Roderick J Clifton-Bligh22,
 * Magdalena Bialas23,
 * Miikka Vikkula24,
 * Gustavo Baretton25,
 * Mauro Papotti10,
 * Gabriella Nesi26,
 * Cécile Badoual13,14,27,
 * Karel Pacak18,
 * Graeme Eisenhofer28,
 * Henri J Timmers20,
 * Felix Beuschlein  ORCID: orcid.org/0000-0001-7826-398419,
 * Jérôme Bertherat29,30,
 * Massimo Mannelli15,31,
 * Mercedes Robledo16,32,
 * Anne-Paule Gimenez-Roqueplo13,14,
 * Winand NM Dinjens2,
 * Esther Korpershoek2 &
 * …
 * Ronald R de Krijger2,33,34 

Show authors

Modern Pathology volume 28, pages 807–821 (2015)Cite this article

 * 10k Accesses

 * 148 Citations

 * 16 Altmetric

 * Metrics details


ABSTRACT

Despite the established role of SDHB/SDHA immunohistochemistry as a valuable
tool to identify patients at risk for familial succinate dehydrogenase-related
pheochromocytoma/paraganglioma syndromes, the reproducibility of the assessment
methods has not as yet been determined. The aim of this study was to investigate
interobserver variability among seven expert endocrine pathologists using a
web-based virtual microscopy approach in a large multicenter
pheochromocytoma/paraganglioma cohort (n=351): (1) 73 SDH mutated, (2) 105
non-SDH mutated, (3) 128 samples without identified SDH-x mutations, and (4) 45
with incomplete SDH molecular genetic analysis. Substantial agreement among all
the reviewers was observed either with a two-tiered classification (SDHB
κ=0.7338; SDHA κ=0.6707) or a three-tiered classification approach (SDHB
κ=0.6543; SDHA κ=0.7516). Consensus was achieved in 315 cases (89.74%) for SDHB
immunohistochemistry and in 348 cases (99.15%) for SDHA immunohistochemistry.
Among the concordant cases, 62 of 69 (~90%) SDHB-/C-/D-/AF2-mutated cases
displayed SDHB immunonegativity and SDHA immunopositivity, 3 of 4 (75%) with
SDHA mutations showed loss of SDHA/SDHB protein expression, whereas 98 of 105
(93%) non-SDH-x-mutated counterparts demonstrated retention of SDHA/SDHB protein
expression. Two SDHD-mutated extra-adrenal paragangliomas were scored as SDHB
immunopositive, whereas 9 of 128 (7%) tumors without identified SDH-x mutations,
6 of 37 (~16%) VHL-mutated, as well as 1 of 21 (~5%) NF1-mutated tumors were
evaluated as SDHB immunonegative. Although 14 out of those 16
SDHB-immunonegative cases were nonmetastatic, an overall significant correlation
between SDHB immunonegativity and malignancy was observed (P=0.00019). We
conclude that SDHB/SDHA immunohistochemistry is a reliable tool to identify
patients with SDH-x mutations with an additional value in the assessment of
genetic variants of unknown significance. If SDH molecular genetic analysis
fails to detect a mutation in SDHB-immunonegative tumor, SDHC promoter
methylation and/or VHL/NF1 testing with the use of targeted next-generation
sequencing is advisable.


MAIN

Pheochromocytomas and paragangliomas are neural crest-derived neuroendocrine
tumors arising from the adrenal medulla and sympathetic/parasympathetic
paraganglia, respectively.1 These carry the highest degree of heritability among
human neoplasms. Germline and/or somatic mutations of at least 18 genes (NF1,
RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, HIF2A, KIF1B, PHD1,
PHD2/EGLN1, FH, HRAS, BAP1, and MEN1) are involved in development of the tumors,
with ∼40% harboring a germline mutation and an additional 25–30% a somatic
mutation.2, 3, 4

Familial succinate dehydrogenase-related pheochromocytoma/paraganglioma
syndromes are caused by SDHA, SDHB, SDHC, SDHD, and SDHAF2 (collectively SDH-x)
mutations and inherited as autosomal dominant traits.4 These syndromes
predispose not only to pheochromocytomas/paragangliomas, but also to
gastrointestinal stromal tumors, renal cell carcinomas, and pituitary
adenomas.5, 6, 7 In the vast majority of succinate dehydrogenase-associated
tumors, there is also loss of SDHB and/or SDHA protein expression that can be
detected by immunohistochemistry.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41 In particular, SDHB-, SDHC-, and SDHD-mutated tumors display SDHB
immunonegativity but SDHA immunoreactivity, whereas SDHA-mutated tumors show
negativity for both SDHB and SDHA immunostainings. Gastrointestinal stromal
tumors and paragangliomas, associated with Carney triad (the syndromic but
nonhereditary association of gastrointestinal stromal tumor, paraganglioma,
pulmonary chondroma, adrenocortical adenoma, and esophageal leiomyoma),4 show
negative staining for SDHB in the absence of SDH-x mutations.29, 40 There is
provisional evidence that Carney triad-related tumors display somatic
hypermethylation of the SDHC promoter locus,42 and therefore negative staining
for SDHB may also identify these cases not found by conventional molecular
testing.

As loss of SDHB/ SDHA expression is predictive of an underlying SDH-x germline
mutation,8, 10, 11, 17, 21, 22, 23, 24, 29, 34, 39 the role of SDHB/SDHA
immunohistochemistry has been underlined as a supplementary approach in
molecular genetic testing especially for pheochromocytomas and paragangliomas.8,
10, 11 As Sanger or targeted next-generation sequencing analysis of all
pheochromocytoma/paraganglioma susceptibility genes is labor intensive and/or
requires clinical molecular diagnostic laboratories,43, 44, 45 it might be
prudent to use immunohistochemistry to identify patients with succinate
dehydrogenase-related pheochromocytoma/paraganglioma syndromes. In addition, the
presence of an SDHB mutation is one of the strongest predictors for both
metastasis and subsequently poor outcome in pheochromocytomas/paragangliomas.4
In this context, it has been proposed that a combination of the GAPP (grading
system for adrenal phaeochromocytoma and paraganglioma) and SDHB
immunohistochemistry might be a valuable aid in the prediction of metastatic
disease,46 further necessitating correct interpretation of SDHB/SDHA
immunostainings.

Given the high prevalence of unsuspected hereditary disease, false-positive as
well as false-negative evaluations of SDHB/SDHA immunostainings can lead to
failure to identify pheochromocytoma/paraganglioma-affected individuals at
increased risk for succinate dehydrogenase-related neoplasia, incorrect
interpretation of the pathogenicity of genetic variants of uncertain
significance, and inappropriate genetic testing. Because studies addressing the
issue of interobserver variation for SDHB/SDHA immunohistochemistry in
pheochromocytomas/paragangliomas are lacking, we assessed interobserver
agreement among practicing expert endocrine pathologists through virtual
microscopy in a large multicenter, multinational cohort of genetically
well-characterized tumors. Accordingly, we examined the validity of SDHB/SDHA
immunohistochemistry to identify patients with succinate dehydrogenase-related
pheochromocytomas/paragangliomas and of SDHB immunohistochemistry as a marker of
malignancy.


MATERIALS AND METHODS


CASE SELECTION

A total of 351 paraganglionic tumors from 333 patients of median age 46 years
(ranging from 5.5 to 84 years; 56% females) were retrieved from 15 specialized
centers from Europe, United States, and Australia: (1) Université catholique de
Louvain, Brussels, Belgium (95 samples from 84 patients), (2) Hôpital Européen
Georges Pompidou, Paris, France (68 samples from 67 patients), (3) University of
Florence, Florence, Italy (40 samples), (4) National Institutes of Health (NIH),
Bethesda, MD, USA (24 samples), (5) Klinikum der Universität München, Munich,
Germany (20 samples), (6) Radboud University Nijmegen Medical Center, Nijmegen,
The Netherlands (18 samples from 17 patients), (7) Instituto Português de
Oncologia de Lisboa Francisco Gentil E.P.E., Lisbon, Portugal (15 samples from
12 patients), (8) Hôpital Cochin, Paris, France (13 samples), (9) Jagiellonian
University Medical College, Krakow, Poland (12 samples), (10) Technische
Universität Dresden, Dresden, Germany (11 samples), (11) San Luigi Gonzaga
Hospital and University of Turin, Turin, Italy (11 samples), (12) Erasmus MC
Cancer Institute, Rotterdam, The Netherlands (10 samples from 8 patients), (13)
University of Sydney, Sydney, Australia (8 samples), (14) Spanish National
Cancer Research Centre (CNIO), Madrid, Spain (5 samples), and (15) Hospital
Universitario San Cecilio, Granada, Spain (1 sample). Clinical and genetic
characteristics of these patients are detailed in Supplementary Tables 1 and 2.
Thirty samples (30 out of 351; 8.54%) were considered malignant (Supplementary
Table 2) as primary tumors and/or recurrences in the presence of metastatic
disease to sites where chromaffin tissue is not normally found4 or as metastases
themselves.

Out of 351 tumor samples, (1) 73 were SDH-x mutated (39 SDHD, 24 SDHB, 4 SDHA, 4
SDHAF2, and 2 SDHC), (2) 105 non-SDH-x mutated (37 VHL, 25 RET, 21 NF1, 8 MAX, 6
HIF2A, 4 TMEM127, and 4 HRAS), (3) 128 wild-type cases (7 head and neck
paragangliomas, 13 extra-adrenal paragangliomas, and 108 pheochromocytomas) that
have been tested negative for mutations and large deletions in the SDH-x genes,
and (4) 45 samples with incomplete SDH-x molecular genetic analysis in terms of
either SDH-x genes or the techniques performed, that is, Sanger sequencing
and/or multiplex ligation-dependent probe amplification. A total of 225 samples
were analyzed at least for 3 pheochromocytoma/paraganglioma susceptibility genes
with 129 and 30 harboring mutations at the germline and somatic level,
respectively (Supplementary Table 1). Based on clinical grounds, 19 tumors were
considered NF1, RET, or VHL mutated (Supplementary Table 1).

None of these tumor samples have been previously published elsewhere in terms of
SDHB/SDHA immunohistochemical investigation and all were anonymously assessed
according to the Proper Secondary Use of Human Tissue code established by the
Dutch Federation of Medical Scientific Societies (http://www.federa.org).
Informed consent was obtained for genetic analysis and access to the clinical
data in accordance with institutional guidelines. The Medical Ethical Committee
of the Erasmus MC approved the study.


SDHB/SDHA IMMUNOHISTOCHEMISTRY

Each case was thoroughly reviewed and representative unstained glass slide(s)
(n= 147) and/or formalin-fixed, paraffin-embedded block(s) (n=204) were selected
and further provided for immunohistochemical analysis within a single research
setting (Department of Pathology, Erasmus MC Cancer Institute, Rotterdam, The
Netherlands) with the following protocol. Slides and formalin-fixed,
paraffin-embedded whole-tissue sections of 4 μm thickness were stained with
commercially available antibodies: (1) mouse monoclonal Ab14715 antibody
(Mitosciences, Abcam, Cambridge, UK; 1:500 dilution) against SDHA and (2) rabbit
polyclonal HPA002868 antibody (Sigma-Aldrich, St Louis, MO, USA; 1:400 dilution)
against SDHB on an automatic Ventana Benchmark Ultra System (Ventana Medical
Systems, Tuscon, AZ, USA) using Ultraview DAB detection system preceded by
heat-induced epitope retrieval with Ventana Cell Conditioning 1 (pH 8.4) at
97 °C for 52 and 92 min, respectively. Diaminobenzidine was used as the
chromogen.


TELEPATHOLOGY APPLICATION

High-resolution, whole-slide images were acquired from 702 SDHB/SDHA
immunostainings using a NanoZoomer Digital Pathology System (Hamamatsu Photonics
KK, Japan) working at a resolution of 0.23 μm/pixel. The immunostainings were
scanned at × 40 magnification and automatically digitized in their proprietary
NanoZoomer Digital Pathology Image file format. A quality control was
subsequently set to ensure good focus. Between August 2012 and December 2013,
digital files were consecutively uploaded in six sets to a server at Erasmus MC
through the standard File transfer Protocol with URL
http://digimic.erasmusmc.nl/, enabling online worldwide viewing through a
virtual microscopy interface (NanoZoomer Digital Pathology.view Viewer Software,
Hamamatsu Photonics KK).


PARTICIPANTS AND INTERPRETATION OF STAINING RESULTS

Seven pathologists, including five who had published on SDHB and/or SDHA
immunohistochemical assessments and two who had dealt with endocrine pathology
on diagnostic and research grounds for many years (AJG, F van N, AST, FT, MV,
XM-G, and RRdeK), received: (1) a word file detailing the context and the
objectives of the project along with an instructory panel of SDHB/SDHA
immunohistochemistry, (2) a Virtual Microscopy (NanoZoomer Digital Pathology)
Manual, (3) the corresponding link providing access to the virtual slides of the
first set of tumors, and (4) a scoring list to be completed during SDHB/SDHA
immunohistochemical evaluations.

All virtual slides were distributed online, reviewed by each observer in a
blinded manner without knowledge of the corresponding clinicopathological and
genetic data or scores assigned by other pathologists and scored as follows: (1)
with regard to SDHB immunohistochemistry: Positive as granular cytoplasmic
staining displaying the same intensity as internal positive control (endothelial
cells, sustentacular cells, lymphocytes); Negative as completely absent staining
in the presence of an internal positive control; Weak diffuse as a cytoplasmic
blush lacking definite granularity contrasting the strong granular staining of
internal positive control; Heterogeneous as granular cytoplasmic staining
combined with a cytoplasmic blush lacking definite granularity or completely
absent staining in the presence of an internal positive control throughout the
same slide; Noninformative as completely absent staining in the absence of an
internal positive control; and (2) with regard to SDHA immunohistochemistry:
Positive as granular cytoplasmic staining displaying the same intensity as
internal positive control (endothelial cells, sustentacular cells, lymphocytes);
Negative as completely absent staining in the presence of an internal positive
control; Heterogeneous as granular cytoplasmic staining combined with a
cytoplasmic blush lacking definite granularity or completely absent staining in
the presence of an internal positive control throughout the same slide;
Noninformative as completely absent staining in the absence of an internal
positive control.

In an effort to simulate widespread adoption of the scoring system as would
occur in community practice, no prescoring consensus meeting was organized. In
order to imitate clinical practice as much as possible for SDHB/SDHA
immunohistochemical interpretations, we selected a large retrospective cohort
comprising SDH-x- and non-SDH-x-mutated paraganglionic tumors with and without
mutations in the remainder pheochromocytoma/paraganglioma-associated genes.


STATISTICAL ANALYSIS

Interobserver agreement was assessed using κ statistics; the strength of the
former was evaluated with criteria previously described by Landis and Koch.47 A
κ-value of <0 indicates less than chance agreement, <0.20 is regarded as slight
agreement, 0.21–0.40 as fair agreement, 0.41–0.60 as moderate agreement,
0.61–0.80 as substantial agreement, 0.81–0.99 as almost perfect agreement, and 1
indicates perfect agreement. A dichotomous classification was used for the
analysis of the pathologists’ evaluations (negative/weak diffuse and positive)
as well as a three-tiered classification approach (negative/weak diffuse,
positive, and heterogeneous). Consensus was defined as agreement at least among
five out of seven pathologists reaching the same interpretation on positive,
negative/weak diffuse, heterogeneous, and noninformative expression for
SDHB/SDHA immunohistochemistry. Discordant evaluation was defined as at least
three observers reporting different SDHB/SDHA expression patterns on the same
slide. In order to capture the performance of SDHB immunohistochemistry as a
predictive tool, we calculated Youden’s J statistic (Youden’s index) per
pathologist either in tumors harboring SDH-x mutations vs non-SDH-x mutations or
in SDH-x-mutated tumors vs counterparts without identified SDH-x mutations. We
used Pearson’s χ2 test to associate (1) SDHB IHC status with biological behavior
(ie, benignancy vs malignancy) taking into consideration only concordant cases
as well as excluding metastases (n=7) and doubled samples (n=6) (Supplementary
Table 2), and (2) SDHD mutations and weak diffuse pattern on SDHB
immunohistochemistry based on a consolidated call from at least four observers.
Two-sided P-values of <0.05 were considered statistically significant.
Statistical analyses were performed using Analyse-it v2.26 (Analyse-it Software,
Leeds, UK).


RESULTS

The interobserver agreement following a two-tiered classification approach (ie,
positive and weak diffuse/negative) ranged from moderate to almost perfect for
SDHB immunohistochemistry and from fair to perfect for SDHA immunohistochemistry
(Table 1). With regard to SDHB immunohistochemistry, the highest agreement was
reached between observers 2 and 3 (κ=0.8593) and the lowest between observers 4
and 7 (κ=0.5318), whereas regarding SDHA immunohistochemistry, the highest
agreement was reached between observers 6 and 2/7 (κ=1.0000) and the lowest
between observers 4 and 5 (κ=0.3542). All agreements were highly significant
(P<0.0001). Substantial agreement among all the reviewers was observed either
with a two-tiered classification (SDHB κ=0.7338; SDHA κ=0.6707) or a
three-tiered classification approach (SDHB κ=0.6543; SDHA κ=0.7516). Notably,
observer 1 as well as observers 3/4/5 did not score any slide as heterogeneous
pattern for SDHB and SDHA immunohistochemistry respectively.

Table 1 Interobserver agreement (κ-values) for SDHA (upper half) and SDHB (lower
half) immunohistochemistry
Full size table

Consensus among pathologists was achieved in 348 cases (99.15%) for SDHA
immunohistochemistry and in 315 cases (89.74%) for SDHB immunohistochemistry,
respectively. Out of 69 tumor samples with SDHB/SDHC/SDHD/SDHAF2 mutations, 62
(89.85%) displayed SDHB immunonegativity and SDHA immunopositivity, whereas 3 of
4 with SDHA mutations (75%) showed loss of SDHA/SDHB protein expression (Figure
1). Two SDHD-mutated extra-adrenal paragangliomas (c.274G>T p.Asp92Tyr and
c.405delC p.Phe136Leufs*32) were scored as SDHB immunopositive by 5 observers
and as immunonegative (weak diffuse) by the other observers (observers 2/5).

Figure 1

SDHA and SDHB immunohistochemistry in pheochromocytomas/paragangliomas endowed
either with SDHA germline mutation displaying loss of SDHA/SDHB protein
expression or with SDHB, SDHC, SDHD, and SDHAF2 germline mutations exhibiting
loss of SDHB, but intact SDHA expression. Note the granular, cytoplasmic
staining for SDHA/SDHB in normal cells of the intratumoral fibrovascular network
that serve as internal positive controls.

Full size image

All tumors harboring RET, TMEM127, HIF2A, and HRAS mutations, 31 of 37
VHL-mutated tumors (83.7%), and 20 of 21 NF1 mutated-tumors (95.2%) displayed
retention of SDHB/SDHA expression (Figure 2). Six benign VHL-mutated
pheochromocytomas (6 out of 37; ~16%) and one malignant NF1-mutated
extra-adrenal paraganglioma (1 out of 21; ~5%) were evaluated as SDHB
immunonegative (VHL: by all observers (3 cases), 6 observers (1 case), and 5
observers (2 cases); NF1: by 6 observers (1 case)) in the absence of SDH-x
mutations in four of these cases (two examined at the germline, one at the
germline and somatic, and one at the somatic level). Data on the exact mutations
were available only in four cases (VHL p.Ser80Asn, p.Arg161*, p.Arg167Gln, and
NF1 p.Trp561*).

Figure 2

Intact SDHB and SDHA protein expression in non-SDH-x-mutated paraganglionic
tumors harboring germline or somatic VHL, RET, NF1, TMEM127, MAX, EPAS1, and
HRAS mutations. Note the granular, cytoplasmic staining for SDHA/SDHB in normal
cells of the intratumoral fibrovascular network that serve as internal positive
controls.

Full size image

In the absence of SDH-x mutations, 119 out of 128 paraganglionic tumors (93%)
were scored as SDHB/SDHA immunopositive, whereas the remainder (9 out of 128;
7%) as SDHB immunonegative/SDHA immunopositive. Clinicopathological and genetic
data of the latter from four independent centers are detailed in Table 2.

Table 2 Clinicopathological and genetic data of patients with
SDHB-immunonegative paraganglionic tumors in the absence of SDH-x mutations
Full size table

Discordant evaluations of SDHB immunohistochemistry were reported in 5 tumors
endowed with SDH-x (SDHD/SDHB/SDHAF2) mutations, 11 VHL- and 2 RET-mutated
tumors, as well as 18 tumors without identified SDH-x mutations, whereas of SDHA
immunohistochemistry concerned 2 SDH-x-mutated tumors (SDHA-/SDHD-) and 1
NF1-mutated tumor. The classification of stainings as ‘noninformative’ and
‘heterogeneous’ represented the major reason for SDHB/SDHA immunohistochemical
discrepancies in the SDH-x-mutated subgroup, whereas the ‘weak diffuse’ category
accounted largely for those in the SDH-x-wild-type and VHL-mutated subsets.

The association between the predicted SDH genetic status and SDHB
immunohistochemistry was investigated for each observer. The sensitivity of this
approach, defined as the percentage of SDH-x mutated tumors that are SDHB
immunonegative, ranged from 83.58 to 98.57% (mean 94.23%). The specificity,
defined as the percentage of either non-SDH-x-mutated tumors or tumors without
identified SDH-x mutations that are SDHB immunopositive, varied between 74.03
and 96.11% (mean 84.35%) as well as 83.06 and 92.91% (mean 86.67%),
respectively. Observer 1 was the best predictor with a Youden’s index of 0.880
and 0.860 (Table 3). A significant correlation was observed between SDHB
immunonegativity and malignancy (P=0.00019). No association could be shown
between the SDHD mutations and the weak diffuse pattern on SDHB
immunohistochemistry (P=0.1490).

Table 3 Associating predicted SDHB IHC status either with SDH-x-mutated vs
non-SDH-x-mutated status (A) or with SDH-x-mutated vs SDH-x-wild-type status
(B)a
Full size table


DISCUSSION

Immunohistochemistry has revolutionized the practice of endocrine pathology
during the last decade. In parallel with recent advances in molecular genetics,
immunohistochemistry has been shown to detect various types of molecular
alterations, that is, BRAF V600E mutation in papillary thyroid carcinomas,48
PTEN mutations in various neoplastic thyroid lesions,49 CTNNB1 mutations in
cribriform-morular variant of papillary thyroid carcinoma, undifferentiated
carcinomas of the thyroid gland and adrenocortical carcinomas,48, 50, 51 TP53
mutations as well as mutations in mismatch repair (MMR) genes such as MLH1,
MSH2, MSH6, and PMS2 in adrenocortical carcinomas,51, 52, 53 HRPT2 mutations in
parathyroid carcinomas and hyperparathyroidism-jaw tumor syndrome-related
adenomas,48, 54 PRKAR1A mutations in Carney complex-associated tumors,55, 56, 57
and SDH-, FH- as well as MAX deleterious-mutations in
pheochromocytomas/paragangliomas.8, 10, 11, 58, 59

Loss of SDHB protein expression is seen in pheochromocytomas/paragangliomas
either harboring a mutation in any of the SDH genes or with somatic
hypermethylation of the SDHC promoter region,42 whereas loss of both SDHB and
SDHA immunoreactivity is demonstrated only in the context of an SDHA mutation.8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 In agreement with previous
studies,8, 10, 11, 17, 18, 19, 20 SDHB-/C-/D- and SDHA-mutated tumors displayed
the aforementioned immunoexpression patterns with SDHAF2-mutated counterparts
showing SDHB immunonegativity and SDHA immunopositivity. Notably, all tumors
harboring RET, TMEM127, HIF2A, and HRAS mutations displayed retention of
SDHB/SDHA expression, whereas six benign VHL-mutated pheochromocytomas and one
malignant NF1-mutated extra-adrenal paraganglioma were evaluated as SDHB
immunonegative. The latter contrasts previous observations in 37
pheochromocytomas/paragangliomas and 14 pheochromocytomas endowed with VHL8, 11
and NF1 mutations,8, 10 respectively. By using a mouse monoclonal (21A11) SDHB
antibody at a low concentration (1 in 1000), Gill et al10 suggested that
VHL-associated tumors could be classified as negative or weak diffuse rather
than positive as demonstrated by a high concentration approach of two SDHB
antibodies.8 In accordance, loss of SDHB protein expression has been recently
displayed in a subset of NF1-mutated paraganglionic tumors (J Favier 2014,
personal communication). The remote possibility of a double mutant, potentially
explaining the SDHB immunonegativity by an additional SDH-x mutation, was ruled
out in four of these seven cases occurring in the VHL- and NF1-deficient
setting.

To further expand earlier observations,8, 11 9 of 128 (7%) tumors without
identified SDH-x mutations were evaluated as SDHB immunonegative (Table 2). Van
Nederveen et al8 and Castelblanco et al11 reported on 9 cases (6 out of 53; 11%
and 3 out of 19; 15.7%) displaying loss of SDHB expression in the absence of
SDHB, SDHC, SDHD, VHL, or RET mutation. Nevertheless, these studies lacked
either SDHA/SDHAF2 genetic testing8, 11 or screening for large-scale SDH-x
deletions11 that may account for higher percentages. Intriguingly, in the
present study, eight SDHB-immunonegative tumors were nonmetastatic in the
absence of SDH-x mutations (Table 2), bearing a close resemblance to the Carney
triad-associated counterparts in terms of SDHB immunohistochemistry and biologic
behavior.4, 29, 60 Because somatic hypermethylation of SDHC was not
investigated, the possibility that the aforementioned tumors represented cases
of Carney triad could not be assessed. Nevertheless, as shown herein, SDHB
immunohistochemical status overall is strongly correlated with the clinical
behavior of pheochromocytoma/paraganglioma, further strengthening the role of
SDHB immunohistochemistry as a prognostic marker.46, 61

Our data reinforce the notion that immunohistochemistry is a valid tool to
identify patients at risk for familial succinate dehydrogenase-related
pheochromocytoma/paraganglioma syndromes, although occasionally this might be
difficult even in a specialized setting (Table 3). Exemplifying the latter, two
extra-adrenal paragangliomas with missense and frameshift SDHD mutations were
scored as SDHB immunopositive by five observers. Similar discrepancy has been
previously reported for an extra-adrenal paraganglioma harboring a nonsense SDHD
mutation (c.14G>A p.Trp5*) in a patient with Carney Stratakis syndrome.31 Given
that the patient additionally developed an SDHB-immunonegative gastrointestinal
stromal tumor31 and that identical missense and nonsense SDHD mutations in other
tumors have led to absence of SDHB expression,5, 8 it is possible that either
the second hit in the SDHD gene in the paraganglioma resulted in an inactive
succinate dehydrogenase complex with preservation of antigenicity or that the
interpretation was erroneous. Of note, every pathologist in the current study
missed at least one SDH-x-related tumor, and these most frequently involved
mutations in SDHD. This suggests SDHD immunohistochemistry as a potential
complementary tool to SDHB immunohistochemistry to identify SDHD-mutated
patients.62 Further adding to those rare familial cases characterized by
disparity between molecular genetic aberrations of a tumor suppressor gene and
retention of protein expression,63 one papillary renal cell carcinomas arising
in a patient with a germline missense SDHC mutation (c.3G>A p.M1I) and harboring
somatic loss of heterozygosity of the SDHC locus paradoxically displayed SDHB
immunopositivity.36 Taken together, SDHB immunohistochemistry and SDH-x genetic
analysis should be viewed as complementary tests. In cases of strong clinical
suspicion, follow-up mutational analysis should be considered despite retention
of SDHB expression.

The good level of reproducibility in the current study may either reflect a high
level of experience with scoring SDHB/SDHA immunostainings among expert
endocrine pathologists or be attributable in part to the fact that very precise
scoring guidelines were provided. Accordingly, it would be essential to provide
such guidelines in clinical reporting templates64 as well as to guide
development of algorithms for computer-assisted diagnostics in a digital
pathology perspective. The classification of stainings as ‘non-informative’ and
‘heterogeneous’ represented the major reason for SDHA/SDHB immunohistochemical
discrepancies in the SDH-x-mutated subgroup, whereas the ‘weak diffuse’ category
accounted largely for inconsistencies in the SDH-x-wild-type and VHL-mutated
subsets. These could be potentially ascribed to (1) technical variability owing
to differences in fixation time, buffered formalin concentrations, and/or age of
the formalin-fixed, paraffin-embedded blocks,10, 11 (2) biological variability,
for example, reduced SDHB protein levels in VHL-mutated paraganglionic tumors,65
or even to (3–4) individual conceptions and experience from specific staining
protocols, as has been shown with immunohistochemistry for MMR proteins.66
Technically suboptimal immunostainings were not unexpectedly encountered given
the fact that provided material was derived from several pathology laboratories,
each following their own fixation and embedding protocols; highlighting the
importance of standardizing preanalytical variables in surgical pathology
specimens.67, 68

In contrast to previous studies10, 11 indicating a stronger correlation of weak
diffuse pattern with SDHD mutations, we could not significantly reinforce this
particular association. Moreover, SDHB and/or SDHA immunohistochemistry may not
always be an all-or-none phenomenon. In particular, two SDHA- and SDHAF2-mutated
tumors displayed a heterogeneous expression pattern (Figures 3 and 4) being
consistent with previous observations concerning SDHB immunohistochemistry in a
pituitary adenoma harboring an SDHD germline mutation.37 Along the same lines,
heterogeneous expression patterns have been reported both with MMR protein
immunohistochemistry in Lynch syndrome and PTEN immunohistochemistry in Cowden
syndrome.49, 69, 70 The biologic nature of heterogeneous tumors in these genetic
contexts is currently unknown.37, 49, 69, 70 Because of potential
misinterpretation of heterogeneous patterns for SDHB and/or SDHA protein loss,
SDH genetic testing is recommended when confronted with such cases.

Figure 3

An extra-adrenal paraganglioma harboring an SDHA (c.1534C>T, p.Arg512*) germline
mutation, metastatic to a paraaortic lymph node, displaying SDHB
immunonegativity (a, c), but a heterogeneous staining pattern for SDHA (b, d–f):
central area (d) convincingly negative for SDHA, peripheral areas (f)
convincingly positive for SDHA, and transitional zones (e) in between exhibiting
cells with intact SDHA expression intermingled with cells with absent SDHA
expression. Three pathologists correctly classified this sample as heterogeneous
for SDHA, with the remainder four observers as positive for SDHA. Note the
granular, cytoplasmic staining for SDHA/SDHB in normal cells of the intratumoral
fibrovascular network that serve as internal positive controls.

Full size image
Figure 4

An SDHAF2-mutated (c.232G>A, p.Gly78Arg) head and neck paraganglioma showing
areas convincingly negative for SDHB and to a lesser extent areas convincingly
positive for SDHB (a). Three pathologists correctly classified this sample as
heterogeneous for SDHB, with the remainder four as negative for SDHB, whereas
all observers scored it as SDHA immunopositive (b). Note the granular,
cytoplasmic staining for SDHB in normal cells of the intratumoral fibrovascular
network that serve as internal positive control.

Full size image

In addition to a comprehensive next-generation sequencing-based strategy for the
analysis of multiple pheochromocytoma/paraganglioma susceptibility genes,43, 44,
45 several algorithms have been proposed as a targeted approach to genetic
testing in clinical practice.8, 71, 72, 73, 74 In this rapidly expanding field,
the importance of assessing the pathogenicity of a ‘variant of unknown
significance’ has become a major and complex problem facing diagnostic
laboratories. Our data further strengthen the role of SDHB/SDHA
immunohistochemistry in determining the functionality of such variants, alone or
in an integrated approach with in silico analysis75, 76 and/or western blot
analysis, succinate dehydrogenase enzymatic assay, and mass spectrometric-based
measurements of ratios of succinate/fumarate and other metabolites.77, 78, 79

In the current study, we conclude that SDHB/SDHA immunohistochemistry represents
a reliable tool to identify patients with SDH-x mutations with an additional
utility to evaluate the pathogenicity of SDH variants of unknown significance in
the new next-generation sequencing era. A heterogeneous SDHB and/or SDHA
immunoexpression pattern has to be followed by SDH molecular genetic testing,
although a SDHB-immunonegative subset of VHL- and NF1-mutated paraganglionic
tumors challenges the issue of specificity for SDHB immunohistochemistry. Hence,
if SDH genetics fails to detect a mutation in SDHB-immunonegative tumor, SDHC
promoter methylation and/or VHL/NF1 testing with the use of targeted
next-generation sequencing is advisable. Our findings highlight the need for
quality assessment programs regarding not only standardized staining protocols,
but also SDHB/SDHA immunohistochemical evaluation procedures. In a prospective
setting, with standardized tissue fixation combined with a locally fine-tuned
immunohistochemical staining protocol, the sensitivity and specificity of the
SDHA/SDHB immunohistochemistry can be improved.


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ACKNOWLEDGEMENTS

This study was supported by the Seventh Framework Programme (FP7/2007–2013)
under grant agreement no. 259735 (ENS@T-Cancer). Genetic analysis of the Belgian
subset was partly supported by the Fonds de la Recherche Scientifique Médicale
(FRSM) convention number 3.4.587.08 F (to AP). AP, SA, and MV acknowledge the
contribution of N Lannoy, A Mendola, and L Evenepoel (Clin. Univ. St-Luc/UCL)
for genetic analysis and F Severino (Clin. Univ. St-Luc) for maintenance of the
database. Besides Professor A Mourin (Clin. Univ. St-Luc/ UCL), they are also
grateful to all pathologists who contributed tumor samples: Professors M Delos
and B Weynand and Drs M-C Nollevaux and C Fervaille (Cl. Un. De Mont-Godinne,
UCL); Drs N Detrembleur, N Blétard, and I Scagnol (CHU Sart-Tilman, Ulg); Drs E
Laterre and G Beniuga (IPG Gosselies); Professor H De Raeve and Dr W Jeuris
(OLV, Aalst); Dr V Duwel (Ziekenhuis KLINA); Drs A Janssen and S Talpe (Clniques
du Sud-Luxembourg, Arlon); Drs C Robrechts and J Bekaert (Imelda Ziekenhuis); Dr
R Duttmann (CHU Brugmann); Dr N de Saint-Aubain (Institut Bordet); and Drs R
Achten and K Wouters (Jessa Ziekenhuis). MM, ER, LC, and GN acknowledge the
contribution of Dr T Ercolino for the genetic analysis of the Italian (Florence)
samples. GE and GB acknowledge the contribution of Drs C Pamporaki, R Därr, S
Richter, and J Brütting for data collection of the German (Dresden) samples. We
thank J Shukla (Erasmus MC Cancer Institute) for her valuable technical
assistance as well as Drs M Versasky and M Gomez Morales (Hospital Universitario
San Cecilio) for providing one SDHD-mutated HNPGL sample.


AUTHOR INFORMATION


AUTHORS AND AFFILIATIONS

 1.  Department of Histopathology, King’s College Hospital, London, UK
     
     Thomas G Papathomas

 2.  Department of Pathology, Erasmus MC Cancer Institute, University Medical
     Center Rotterdam, Rotterdam, The Netherlands
     
     Thomas G Papathomas, Lindsey Oudijk, Winand NM Dinjens, Esther
     Korpershoek & Ronald R de Krijger

 3.  Institut de Recherche Expérimentale et Clinique and Division of Cardiology,
     Pole of Cardiovascular Research, Cliniques Universitaires Saint-Luc,
     Université Catholique de Louvain, Brussels, Belgium
     
     Alexandre Persu

 4.  Department of Anatomical Pathology, Royal North Shore Hospital, St
     Leonards, NSW, Australia
     
     Anthony J Gill

 5.  Cancer Diagnosis and Pathology Research Group, Kolling Institute of Medical
     Research, University of Sydney, Sydney, NSW, Australia
     
     Anthony J Gill

 6.  Laboratory for Pathology, PAL Dordrecht, Dordrecht, The Netherlands
     
     Francien van Nederveen

 7.  Department of Pathology and Laboratory Medicine, Tufts Medical Center,
     Tufts University School of Medicine, Boston, MA, USA
     
     Arthur S Tischler

 8.  Institut National de la Santé et de la Recherche Médicale U1016, Institut
     Cochin, Centre National de la Recherche Scientifique UMR8104, Université
     Paris Descartes, Sorbonne Paris Cité, Rare Adrenal Cancer Network COMETE,
     Paris, France
     
     Frédérique Tissier & Rosella Libe

 9.  Department of Pathology, Hôpital Pitié-Salpêtrière, Université Pierre et
     Marie Curie, Paris, France
     
     Frédérique Tissier

 10. Department of Oncology, University of Turin at San Luigi Hospital, Turin,
     Italy
     
     Marco Volante & Mauro Papotti

 11. Department of Pathology and Molecular Genetics and Research Laboratory,
     Hospital Universitari Arnau de Vilanova, IRBLLEIDA, University of Lleida,
     Lleida, Spain
     
     Xavier Matias-Guiu

 12. Department of Medical Oncology, Erasmus MC Cancer Institute, University
     Medical Center Rotterdam, Rotterdam, The Netherlands
     
     Marcel Smid

 13. Paris-Centre de Recherche Cardiovasculaire (PARCC), Inserm UMR970, Hôpital
     Européen Georges Pompidou, Paris, France
     
     Judith Favier, Cécile Badoual & Anne-Paule Gimenez-Roqueplo

 14. Université Paris Descartes, Faculté de Médecine, Paris Cité Sorbonne,
     France
     
     Judith Favier, Cécile Badoual & Anne-Paule Gimenez-Roqueplo

 15. Department of Experimental and Clinical Biomedical Sciences, Endocrinology
     Unit, University of Florence, Florence, Italy
     
     Elena Rapizzi, Letizia Canu & Massimo Mannelli

 16. Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre
     (CNIO), Madrid, Spain
     
     Maria Currás-Freixes & Mercedes Robledo

 17. Department of Pathology, Cliniques Universitaires Saint-Luc, Université
     catholique de Louvain, Institut de Recherche Expérimentale et Clinique,
     Brussels, Belgium
     
     Selda Aydin

 18. Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver
     National Institute of Child Health and Human Development, National
     Institutes of Health, Bethesda, MD, USA
     
     Thanh Huynh & Karel Pacak

 19. Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Klinikum
     der Universität München, Munich, Germany
     
     Urs Lichtenauer & Felix Beuschlein

 20. Department of Internal Medicine, Section of Endocrinology, Radboud
     University Medical Centre, Nijmegen, The Netherlands
     
     Anouk van Berkel & Henri J Timmers

 21. Unidade de Investigação em Patobiologia Molecular (UIPM), Instituto
     Português de Oncologia de Lisboa Francisco Gentil, Lisbon, Portugal
     
     Rita Domingues

 22. Cancer Genetics, Kolling Institute of Medical Research, Royal North Shore
     Hospital, University of Sydney, Sydney, NSW, Australia
     
     Roderick J Clifton-Bligh

 23. Department of Pathomorphology, Jagiellonian University Medical College,
     Krakow, Poland
     
     Magdalena Bialas

 24. Laboratory of Human Molecular Genetics, de Duve Institute, Université
     Catholique de Louvain, Brussels, Belgium
     
     Miikka Vikkula

 25. Department of Pathology, Technische Universität Dresden, Dresden, Germany
     
     Gustavo Baretton

 26. Division of Pathological Anatomy, University of Florence, Florence, Italy
     
     Gabriella Nesi

 27. Service d'Anatomie Pathologique, Hôpital Européen Georges-Pompidou,
     Assistance Publique Hôpitaux de Paris (AP-HP), Paris, France
     
     Cécile Badoual

 28. Institute of Clinical Chemistry and Laboratory Medicine and Department of
     Medicine III, University Hospital, Technische Universität Dresden, Dresden,
     Germany
     
     Graeme Eisenhofer

 29. Institut Cochin, Université Paris Descartes, INSERM U1016, CNRS UMR8104,
     Paris, France
     
     Jérôme Bertherat

 30. Department of Endocrinology, Referral Center for Rare Adrenal Diseases,
     Assistance Publique Hôpitaux de Paris, Hôpital Cochin, Paris, France
     
     Jérôme Bertherat

 31. Istituto Toscano Tumori (ITT), Florence, Italy
     
     Massimo Mannelli

 32. Centre for Biomedical Network Research on Rare Diseases (CIBERER), Madrid,
     Spain
     
     Mercedes Robledo

 33. Department of Pathology, Reinier de Graaf Hospital, Delft, The Netherlands
     
     Ronald R de Krijger

 34. Department of Pathology, University Medical Center Utrecht, Princess Maxima
     Center for Pediatric Oncology, Utrecht, The Netherlands
     
     Ronald R de Krijger

Authors
 1.  Thomas G Papathomas
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 2.  Lindsey Oudijk
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 5.  Francien van Nederveen
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 6.  Arthur S Tischler
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 7.  Frédérique Tissier
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 8.  Marco Volante
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 9.  Xavier Matias-Guiu
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 18. Anouk van Berkel
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 20. Rita Domingues
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 30. Henri J Timmers
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 31. Felix Beuschlein
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 33. Massimo Mannelli
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 34. Mercedes Robledo
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 35. Anne-Paule Gimenez-Roqueplo
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 36. Winand NM Dinjens
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 37. Esther Korpershoek
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 38. Ronald R de Krijger
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CORRESPONDING AUTHOR

Correspondence to Thomas G Papathomas.


ETHICS DECLARATIONS


COMPETING INTERESTS

The authors declare no conflict of interest.


ADDITIONAL INFORMATION

Supplementary Information accompanies the paper on Modern Pathology website


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SUPPLEMENTARY TABLE 1 (XLS 91 KB)


SUPPLEMENTARY TABLE 2 (XLS 19 KB)


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CITE THIS ARTICLE

Papathomas, T., Oudijk, L., Persu, A. et al. SDHB/SDHA immunohistochemistry in
pheochromocytomas and paragangliomas: a multicenter interobserver variation
analysis using virtual microscopy: a Multinational Study of the European Network
for the Study of Adrenal Tumors (ENS@T). Mod Pathol 28, 807–821 (2015).
https://doi.org/10.1038/modpathol.2015.41

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 * Received: 26 October 2014

 * Revised: 10 January 2015

 * Accepted: 10 January 2015

 * Published: 27 February 2015

 * Issue Date: June 2015

 * DOI: https://doi.org/10.1038/modpathol.2015.41


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 * Adrenal tumours


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 * Abstract
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 * Discussion
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Modern Pathology (Mod Pathol) ISSN 1530-0285 (online) ISSN 0893-3952 (print)


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