ORIGINAL
E n d o c r i n e
ARTICLE
R e s e a r c h
Krebs Cycle Metabolite Profiling for Identification
and Stratification of Pheochromocytomas/
Paragangliomas due to Succinate Dehydrogenase
Deficiency
Susan Richter, Mirko Peitzsch, Elena Rapizzi, Jacques W. Lenders, Nan Qin,
Aguirre A. de Cubas, Francesca Schiavi, Jyotsna U. Rao, Felix Beuschlein,
Marcus Quinkler, Henri J. Timmers, Giuseppe Opocher, Massimo Mannelli,
Karel Pacak, Mercedes Robledo, and Graeme Eisenhofer*
Context: Mutations of succinate dehydrogenase A/B/C/D genes (SDHx) increase susceptibility to
development of pheochromocytomas and paragangliomas (PPGLs), with particularly high rates of
malignancy associated with SDHB mutations.
Objective: We assessed whether altered succinate dehydrogenase product-precursor relationships,
manifested by differences in tumor ratios of succinate to fumarate or other metabolites, might aid
in identifying and stratifying patients with SDHx mutations.
Design, Setting, and Patients: PPGL tumor specimens from 233 patients, including 45 with SDHx
mutations, were provided from eight tertiary referral centers for mass spectrometric analyses of
Krebs cycle metabolites.
Main Outcome Measure: Diagnostic performance of the succinate:fumarate ratio for identification of pathogenic SDHx mutations.
Results: SDH-deficient PPGLs were characterized by 25-fold higher succinate and 80% lower fumarate, cis-aconitate, and isocitrate tissue levels than PPGLs without SDHx mutations. Receiveroperating characteristic curves for use of ratios of succinate to fumarate or to cis-aconitate and
isocitrate to identify SDHx mutations indicated areas under curves of 0.94 to 0.96; an optimal
cut-off of 97.7 for the succinate:fumarate ratio provided a diagnostic sensitivity of 93% at a
specificity of 97% to identify SDHX-mutated PPGLs. Succinate:fumarate ratios were higher in both
SDHB-mutated and metastatic tumors than in those due to SDHD/C mutations or without
metastases.
Conclusions: Mass spectrometric-based measurements of ratios of succinate:fumarate and other
metabolites in PPGLs offer a useful method to identify patients for testing of SDHx mutations, with
additional utility to quantitatively assess functionality of mutations and metabolic factors responsible for malignant risk. (J Clin Endocrinol Metab 99: 3903–3911, 2014)
P
heochromocytomas and paragangliomas (PPGLs) are
adrenal and extra-adrenal tumors of neural crest origin. At least one-third of cases are explained by germline
mutations in at least ten tumor-susceptibility genes (1):
neurofibromatosis type 1 (NF1); rearranged during transfection (RET) protooncogene, transmembrane protein
127 (TMEM127); myc-associated factor X (MAX); von
Hippel-Lindau tumor suppressor (VHL) or one of the
genes for succinate dehydrogenase subunits (SDHA, B, C,
D, AF2).
PPGLs due to mutations in SDHB are predominantly
extra-adrenal with low catecholamine contents and asso-
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received April 23, 2014. Accepted June 30, 2014.
First Published Online July 11, 2014
* Author Affiliations are shown at the bottom of the next page.
Abbreviations: FFPE, formalin-fixed paraffin-embedded; HNP, head and neck paraganglioma; MAX, myc-associated factor X; NF1, neurofibromatosis type 1; PPGLs, pheochromocytomas and paragangliomas; RET, rearranged during transfection; ROC, receiver-operating characteristic; TMEM127, transmembrane protein 127; VHL, von Hippel-Lindau
tumor suppressor.
doi: 10.1210/jc.2014-2151
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ciated with high risk for metastatic disease (2, 3). Screening for SDHx-related mutations is therefore important for
identifying patients and family members at risk for developing malignancy, additional PPGLs as well as other types
of tumors that also result from SDHx mutations. The latter are now established to include gastrointestinal stromal
tumors and renal cell carcinomas (4, 5). The rationale is
that earlier detection of tumors through screening of identified patients should result in improved therapeutic outcome and reduced risk of malignancy.
Identification of patients with PPGLs resulting from
SDHx mutations can be facilitated by considerations of
patterns of biochemical parameters and immunohistochemical staining for SDHB protein in resected tumor material (6, 7). With the advent of next-generation sequencing, such triaging for targeted genetic testing may become
obsolete (8). Nevertheless, next-generation sequencing
does not allow detection of SDHx gene deletions particularly important to consider in patients with PPGLs (9,
10). Also as outlined elsewhere (11), broad nonselective
screening by next-generation sequencing has other potential limitations. A particularly major challenge concerns
identification of gene variants that are pathogenic among
a larger proportion of variants of uncertain significance.
Gene prediction tools are used in this context, but the ideal
method is assignment of functionality using quantitative
readouts.
As demonstrated for myeloid leukemia and gliomas due
to mutations of isocitrate dehydrogenase, measurements
of metabolites of the enzyme can provide especially useful
biomarkers of disease and quantitative tools for assessment of functionality and disease stratification (12–14).
Assessment of Krebs cycle metabolism is, however, not
only of interest in leukemia and gliomas, but also in many
other neoplasms including PPGLs (15, 16). In particular,
measurements of tumor tissue levels of succinate and fumarate, the respective substrate and metabolite of succinate dehydrogenase, have now been shown in a proof-ofprinciple pilot study to offer a potentially useful tool for
identification of patients with SDHx mutations (17). Utility of the succinate to glutamate ratio for the same purpose
has also been demonstrated in another small patient cohort (18).
The present study extends the above preliminary findings to a much larger cohort of 233 patients with PPGLs
J Clin Endocrinol Metab, October 2014, 99(10):3903–3911
in whom tumor tissue levels of succinate, fumarate and
other Krebs cycle metabolites were measured using a
novel, rapid, and simple liquid chromatographic tandem
mass spectrometric method. The primary objective of the
study was to assess utility of the succinate:fumarate ratio
for identification of patients with SDHx mutations. For
this we used a training set of tumor samples from 49 patients to establish diagnostic cut-off values, followed by a
blinded validation series of 184 samples to establish diagnostic efficacy. For the secondary objective, both sets of
data were employed to establish utility for disease stratification according to the specific gene inactivated as well
as tumor location and presence of metastatic disease.
Materials and Methods
Patients and tumor procurement
PPGLs were collected from 233 patients (Table 1) undergoing
surgical resection of primary tumors. Tumor procurement was
approved under Intramural Review Board protocols at each participating center. All tumor specimens were provided as frozen
fragments (10 –50 mg). Fifteen cases were also available as formalin-fixed paraffin-embedded (FFPE) specimens to assess utility of such samples for metabolite analyses. As a first step, 50
frozen specimens from 49 patients with known mutational status
were analyzed as a training set. The second step involved provision of 184 tumor samples as a validation set. These latter
specimens were provided blinded to their mutational status.
Krebs cycle metabolites were analyzed and an interpretation concerning the likelihood of an SDHx mutation was provided back
to the centers. The primary study endpoint was the evaluation of
diagnostic sensitivity and specificity using the succinate:fumarate ratio to define SDHx mutational status.
Measurements of tissue metabolites
Processed tissues from primary tumors were analyzed by ultrahigh pressure liquid chromatography with tandem mass spectrometry (UPHPLC-MS/MS). Fresh frozen tumor tissue (5–10
mg) was homogenized in 500 ␮L LC/MS grade methanol containing the internal standard mixture (Supplemental Table 1) by
vortexing with a metal bead for 2 min. To achieve separation of
insoluble debris, homogenates were centrifuged at 2000xg for 5
min at 4°C. Supernatants were dried using a speed vac concentrator (Thermo Scientific) and stored at ⫺80°C. On the day of
analysis, residues were resuspended in mobile phase and cleared
with a 0.2 ␮m centrifugal filter. FFPE tissue was sectioned, and
metabolites were extracted from two slices of 50 ␮m thickness
using the method of Kelly et al (19).
UHPLC-MS/MS was performed on the same instrumentation
Institute of Clinical Chemistry and Laboratory Medicine (S.R., M.P., N.Q., G.E.), University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden,
Fetscherstrasse 74, 01307 Dresden, Germany; Department of Experimental and Clinical Biomedical Sciences “Mario Serio” (E.R., M.M.), University of Florence and Istituto Toscano Tumori,
Viale Pieraccini 6, 50139 Florence, Italy; Department of Medicine (J.W.L., J.U.R., H.J.T.), Radboud University Nijmegen Medical Centre, Geert Grooteplein Zuid 8, 6525GA, Nijmegen, The
Netherlands; Department of Medicine III (J.W.L., G.E.), University Hospital Dresden, Fetscherstrasse 74, 01307 Dresden, Germany; Hereditary Endocrine Cancer Group (A.A.C., M.R.), CNIO,
Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) (A.A.C., M.R.), C/Melchor Fernández Almagro 3, 28029 Madrid, Spain; Veneto Institute of
Oncology IRCCS (F.S., G.O.), Via Gattamelata 64, 35128 Padova, Italy; Medizinische Klinik and Poliklinik IV (F.B.), Ludwig-Maximilians-Universität München, Ziemssenstrasse 1, D-80336
Munich, Germany; Clinical Endocrinology (M.Q.), Campus Mitte, University Hospital Charité, Charitéplatz 1, 10117, Berlin, Germany; Eunice Kennedy Shriver National Institute of Child
Health and Human Development (K.P.), National Institutes of Health, 10 Center Drive, MSC-1109, Bethesda, Maryland 20892-1109
doi: 10.1210/jc.2014-2151
Table 1.
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Patient Demographics and Clinical Characteristics
Germline
Mutation
Training set
SDHB
SDHD
VHL
RET
NF1
None
Validation set
SDHB
SDHD
SDHC
VHL
RET
NF1
TMEM127
Max
None
No. of
Patients
Sex
(M/F)
Age Range
(y; mean)
Tumor Location
(A/E/HN)
Metastatic
Disease
8
3
3
11
8
16
4/4
3/0
1/2
6/5
7/1
7/9
15– 46; 33
32– 46; 39
10 –32; 24
31– 67; 44
17–59; 42
18 –76; 46
1/7/0
1/1/1
2/1/0
11/0/0
8/0/0
12/3a/0 (1U)
4
0
0
0
0
1
16
16
2
8
19
6
3
1
113
9/7
6/10
1/1
6/2
7/12
2/4
1/2
0/1
62/51
14 –57; 34
12–76; 37
16 – 60; 38
11– 43; 22
18 – 65; 37
38 –76; 51
21–54; 36
46
13–78; 50
2/11/3
1/1/14
0/1/1
7/1/0
19/0/0
6/0/0
3/0/0
1/0/0
86/21/6
4
1
0
0
0
0
0
1
9
Abbreviations: U, unknown; A, adrenal; E, extra-adrenal (thoracic and abdominal); HN, head and neck.
a
For one patient, two simultaneously occurring extra-adrenal tumors were analysed.
described for our routine diagnostic measurements of plasma
and urinary normetanephrine, metanephrine, and methoxytyramine (20, 21). This included an Acquity UHPLC system (Waters), equipped with a binary pump manager, a sample manager
and a column manager, coupled to an API QTRAP 5500 triple
quadrupole mass spectrometer (AB Sciex). A Waters Acquity
UPLC® HSS T3 column (1.8 ␮m, 2.1 ⫻ 100 mm) was used for
chromatographic separation. Mobile phases consisted of 0.2%
formic acid in water (A) and 0.2% formic acid in acetonitrile (B)
set at a flow rate of 0.459 mL/min. For each injection an initial
gradient of 5% mobile phase B for 0.37 min was increased to
30% at 4.87 min and 100% at 5.37 min; after column washing
with 100% B for 0.5 min, the column was re-equilibrated with
5% mobile phase B ready for the next injection. Targeted analyses were performed in multiple reaction monitoring scan mode
with use of negative electrospray ionization, as described elsewhere (22). Multiple reaction monitoring transitions for quantification and qualification are listed in Supplemental Table 2.
Interassay variation was established by measuring tissue aliquots of two different tumors at different days with one tumor
showing moderate levels of succinate (QC1) and the other one
having a high succinate concentration (QC2). Interassay coefficients of variation were determined at 6% in QC1 and 4.2% in
QC2 for succinate, and 6.6% and 23.8% for fumarate, respectively. Interassay variations for all metabolites are recorded in
Supplemental Table 1.
Genetic characterization
Genetic testing was performed to confirm or exclude the presence of germline mutations in SDHx genes. With the exception
of patients in whom a germline or somatic mutation in another
PPGL susceptibility gene (RET, NF1, TMEM127, MAX, VHL,
HIF2A) had previously been identified, all patients were tested
for SDHAF1, SDHAF2, SDHA, SDHB, SDHC or SDHD point
mutations by automated sequencing, with detection of gross deletions by multiplex ligation-dependent probe amplification
analysis. Diagnosis of NF1 was based on clinical presentation.
Genetic testing for RET, TMEM127, MAX and VHL mutations
was performed in 70% of the cases. PCR conditions and primers
are available on request. The Alamut mutation interpretation
software (http://www.interactive-biosoftware.com) was used to
assess pathogenicity of previously unidentified variants.
Follow-up testing
Follow-up testing, utilizing additional tumor specimens and
samples of germline DNA, was carried out in cases where tumor
succinate:fumarate ratios indicated either false-negative results
(ratios below the cut-off in patients with SDHx mutations) or
false-positive results (ratios above the cut-off in patients without
SDHx mutations), this according to a written study plan outlined
to all investigators prior to their contributions of patient specimens for the validation series. Genetic testing in patients with
false-positive results included evaluation of tumor tissues for
somatic mutations of all SDHx genes as well as of SDHAF2 and
SDHAF1. Loss of heterozygosity studies were also conducted
mainly directed to false-negative results. For both false-positive
and false-negative results, tissue samples were re-examined for
metabolite levels, SDH enzyme activity, and protein content of
SDHB. SDH activities were measured in tissue homogenates, and
western blots were performed as described previously (23). Densitometry was undertaken using Chemidoc software.
Statistical analysis
Statistical analysis was performed using SigmaPlot 12.0. The
differences between two groups were analyzed by a t-test, when
data were distributed normally or alternatively by rank sum test.
Comparisons between multiple groups were undertaken by oneway ANOVA on ranks. Logistic regression was used to establish
receiver-operating characteristic (ROC) curves, which were used
to identify the optimal cut-off values for discriminating PPGLs
with SDHx-related mutations from others. Sensitivity was calculated by dividing the number of true positives by the sum of
true positives and false negatives. Specificity was estimated by
dividing the number of true negatives by the sum of true negatives
and false positives. Values are provided as mean ⫾ SEM.
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Results
Succinate to fumarate ratios
Succinate:fumarate ratios for PPGLs of the training set
were 454-fold higher (P ⬍ .001) in SDHx-mutated than
other tumors, with values spanning a range of two orders
of magnitude (Figure 1a). Using ROC curve analysis a
cut-off of 97.7 for the succinate:fumarate ratio was determined to provide 100% sensitivity and 97.3% specificity
(Supplemental Figure 1). In three paraganglial specimens,
two of which were from separate extra-adrenal tumors
from the same patient, elevated succinate:fumarate ratios
were detected, but no germline SDHx mutation was noted
(two false-positives in the training set).
Succinate:fumarate ratios for PPGLs of the validation
set were also considerably higher (P ⬍ .001) in SDHxmutated vs other PPGLs (Figure 1b), but the 94-fold difference was smaller than that for the training set. This
smaller difference reflected five specimens with ratios
above the cut-off, but no SDHx germline mutation (five
false-positives in the validation set) and a further three
specimens from patients with confirmed SDHx germline
mutations, but with tumor succinate:fumarate ratios below the cut-off (three false-negatives). For two samples, no
interpretation was provided back to contributing investigators due to findings of high tissue succinate levels with
unusual out of range values for fumarate. These samples
were thus not included in the final analysis.
Follow-up testing
Follow-up genetic testing confirmed the SDHx germline
mutation status in all three patients with false-negative succinate:fumarate ratios (Supplemental Table 3). In all except
J Clin Endocrinol Metab, October 2014, 99(10):3903–3911
one false-positive case, exhaustive testing of SDHx genes in
tumor samples failed to reveal any additional genetic variant
that could explain the high tumor succinate:fumarate ratios.
That one case among the seven with false-positive succinate:
fumarate ratios involved a patient in whom testing of the
head and neck paraganglioma (HNP) revealed a somatic
SDHB mutation (c.380T ⬎ G) confirmed at two independent
centers. That mutation was predicted to be deleterious and
the patient was transferred from a false- to a true-positive
category (six false-positives in total).
All three patients with false-negative tumor succinate:
fumarate ratios had HNPs (Supplemental Table 3). SDH
enzyme activity and western blot analyses indicated results
consistent with mutation-status in all three patients. Specifically, all SDHx-mutated tumors returning false-negative
results for tumor succinate:fumarate ratios had reduced levels of SDH activity (33 ⫾ 6%) compared to wild-type controls (93 ⫾ 6%); tumor tissue also showed reduced SDHB
protein to 36 ⫾ 2% of controls by western blot.
Among the patients with false-positive elevations of
succinate:fumarate ratios, only one presented with an adrenal tumor, the others with paragangliomas (Supplemental Table 3). SDH western blot and activity measurements
supported the findings of high tumor tissue succinate:fumarate ratios in four of the six patients with false-positive
results despite lack of evidence for SDHx mutations.
Profiling of Krebs cycle metabolites
Most Krebs cycle metabolites showed significant differences between tumors with and without SDHx mutations
(Figure 2). Succinate was 25-fold higher in PPGLs with
SDHx mutations, whereas fumarate, citrate, cis-aconitate
Figure 1. Box and whisker plots of succinate:fumarate-ratios for PPGLs analyzed in the training (A) and validation set (B). Boxes span
the 25–75 percentile and whiskers span the 10 –90 percentile. Cut-off, determined by ROC curve analysis of the training set (97.7), is
marked as a dashed line. Black circles indicate samples with values below the cut-off for SDHx tumors or above the cut-off for nonSDHx PPGLs. Two samples were excluded from the analysis of the validation set due to unusually high fumarate and malate values.
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Figure 2. Box and whisker plots comparing metabolite levels between 45 PPGLs with SDHx mutations and 189 other PPGLs. Boxes span
the 25–75 percentile and whiskers span the 10 –90 percentile, with outliers as points. Y-axes indicate tissue levels of metabolites in ng/
mg tissue. Significance was established by the Mann-Whitney Rank sum test.
and isocitrate were, respectively, 80%, 60%, 82%, and 80%
lower. No significant differences between groups were observed for ␣-ketoglutarate, malate, pyruvate, and lactate.
Analysis of subcomponents of the data set showed that
PPGLs due to VHL mutations (germline or somatic), similar to those due to SDHx mutations, also contain less (P ⬍
.05) fumarate, citrate, cis-aconitate, and isocitrate com-
pared to tumors harboring RET, NF1 or TMEM127 mutations (Supplemental Figure 2, b-e). Succinate, however,
was only elevated in tumors with SDH inactivation (Supplemental Figure 2a). Pyruvate was slightly higher in
SDHx and VHL tumors compared to PPGLs with NF1,
RET or TMEM127 mutations, but significance was only
reached for VHL tumors (Supplemental Figure 2f).
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J Clin Endocrinol Metab, October 2014, 99(10):3903–3911
SDHx mutational status, malignancy, and tumor
location
Tumor succinate levels were similar among tumors due
to SDHB and SDHC/SDHD mutations; however, fumarate levels were lower and succinate:fumarate ratios higher
(P ⱕ .01) in SDHB compared to SDHC/D tumors (Figure
3a). Among tumors due to SDHx mutations, fumarate was
lower (P ⫽ .007) and succinate:fumarate ratios higher
(P ⫽ .022) in those associated with metastatic disease than
in those without metastatic disease (Figure 3b). Tumors at
head and neck locations had higher (P ⬍ .001) fumarate
levels and lower (P ⬍ .001) succinate:fumarate ratios compared to those at adrenal, abdominal or thoracic locations
(Figure 3c).
Succinate:fumarate ratio determined in FFPE tissue
In a subset of samples, direct comparison of the succinate:fumarate ratios between fresh-frozen and FFPE tissue
showed similar values for non-SDHx tumors (Supplemental Figure 3). Five of six SDHx-related tumors had succinate:fumarate ratios above the cut-off for fresh-frozen
tissue.
Identification of SDHx mutations based on Krebs
cycle metabolite levels
For all tumors in the validation set considered together,
diagnostic sensitivity and specificity, respectively, reached
90.9% and 97.3%; however, when tumors were separated
according to location, sensitivity increased to 100% for
adrenal, abdominal, and thoracic PPGLs (Table 2). For
head and neck tumors, sensitivity was 83.3% and specificity 80.0%.
For the succinate:fumarate ratio, an area under the
ROC curve of 0.94 was calculated (Figure 4a) with an
overall sensitivity for the combined training and validation set of 93.2% and specificity of 96.8%. Succinate to
citrate, succinate to cis-aconitate and succinate to isocitrate ratios also provided high diagnostic performance
with respective areas under ROC curves of 0.94, 0.95 and
0.96 (Figure 4b).
Discussion
The present study is the first comprehensive analysis of a
large tumor set from multiple centers establishing the validity of measuring succinate:fumarate ratios for both
identifying PPGLs due to SDHx mutations and quantitatively confirming functionality of identified mutations.
Differences in Krebs cycle metabolite profiles according to
driver mutation, metastasis and tumor location indicate
further potential as a quantitative method for disease
stratification.
Figure 3. Box and whisker plots comparing succinate and
fumarate levels and succinate:fumarate ratios between
subgroups of SDHx-related PPGLs (45 tumors in total). (A) 24
PPGLs with SDHB vs 21 with SDHC/D mutations; (B) 9 PPGLs with
vs 36 without metastases; (C) adrenal (PHEO, 5) vs abdominal or
thoracic tumor location (PGL, 21) vs head and neck
paragangliomas (HNP, 19). Boxes span the 25–75 percentile and
whiskers span the 10 –90 percentile, with outliers as points.
Significance was established by Mann-Whitney Rank sum test or
ANOVA on Ranks/Dunn’s test (asterisks, P ⬍ .05).
With an area under the ROC curve of 0.94 and overall
diagnostic sensitivity of 93% and specificity of 97%, use
of tumor tissue succinate:fumarate ratios to identify presence of SDHx mutations is in par with the diagnostic performance of many other tests used in the routine labora-
doi: 10.1210/jc.2014-2151
Table 2.
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Diagnostic Sensitivity and Specificity for Succinate:Fumarate Ratios to Identify SDHx Mutations
Validation set
Sensitivity (%)
Specificity (%)
Training and validation set
Sensitivity (%)
Specificity (%)
All PPGLs
PPGLs Excl. HNP
HNP
90.9 (30/33)
97.3 (145/149)
100 (15/15)
97.9 (141/144)
83.3 (15/18)
80.0 (4/5)
93.2 (41/44)
96.8 (181/187)
100 (25/25)
97.3 (177/182)
84.2 (16/19)
80.0 (4/5)
Analysis excluded two SDHx tumors with low succinate:fumarate ratios, which are the result of extremely high fumarate values. These samples also
showed high, for SDHx mutations typical succinate levels.
Abbreviation: HNP, head and neck paraganglioma.
tory. Moreover with the exclusion of HNPs, the diagnostic
sensitivity of succinate:fumarate ratios to identify tumors
due to SDHx mutations increased to 100%. Thus, for
thoracic, abdominal and adrenal tumors, a succinate:fumarate ratio below the cut-off reliably excludes an SDHx
mutation. HNPs are biochemically different to other
PPGLs, since these tumors mostly lack catecholamine production (24). Intensity of the succinate to fumarate signal
is also lower in SDHx-mutated HNPs than in other SDHxmutated PPGLs, which might reflect differences in the homogeneity of chromaffin tumor cell populations and overall tumor tissue loss of SDH.
With a specificity of 97% the method provides a high
level of confidence that patients with positive results do in
fact harbor an SDHx mutation. Of interest, among the
patients with false-positive elevations of succinate:fumarate ratios most had other evidence pointing to loss of SDH
function. Despite exhaustive follow-up mutation analysis
no explanation was found except for one patient with a
somatic SDHB mutation. Possibly chromosomal translocations, gene methylation, or mutations in promoter regions of SDHx genes may provide explanations. Further
unexplored possibilities include mutations in genes influencing the functionality of the SDH enzyme complex or
metabolic flux through the Krebs cycle. In a subgroup of
HNPs from patients without SDHx germline mutations,
signaling of the HIF-1alpha/miRNA-210 axis resulted in
decreased iron-sulfur cluster scaffold protein, which is suspected to influence SDH protein stability (25). Furthermore, substantial increases in succinate accumulation
have been demonstrated through processes involving increased import and metabolism of glutamine (26). Another study identified the mitochondrial chaperone
TRAP1 as an inhibitor of SDH also leading to increases in
succinate levels (27).
In addition to identification and quantitative functional characterization of SDHx mutations, profiling of
Krebs cycle metabolites may also be useful for disease
stratification, this following from the findings of Letouze
et al that increased tumor levels of succinate lead to DNA
hypermethylation as a critical tumorigenic mechanism
(28). In that study stronger DNA hypermethylation and
the more aggressive nature of PPGLs due to SDHB than
SDHD mutations was hypothesized to be linked to differences in tumor succinate levels. Our findings of higher
succinate to fumarate ratios in PPGLs due to mutations of
SDHB than due to mutations of SDHC/D supports the
suggestion by Letouze et al of stronger functional effects of
the former than of the latter mutations. The higher tumor tissue succinate:fumarate ratios in PPGLs associated with metastases than in those
without metastatic involvement further supports the possibility that these
measurements might have prognostic
value for assessing likelihood of malignancy. Further studies are required,
however, to clarify any confounding
influence of tumor location.
Increased succinate levels in SDHx
mutated tumors have been previously
described in small sets of PPGLs (28 –
Figure 4. ROC curves for identification of SDHx mutations according to succinate to
30) and cell culture models (31, 32).
fumarate ratios in panel A, and succinate to citrate, succinate to cis-aconitate, and
Our findings of low fumarate in SDHx
succinate to isocitrate in panel B. ROC curves are derived from both training and
mutated compared to other tumors
validation set.
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are also consistent with studies in SDH-deficient yeast
strains (32). In addition to the above changes, we also
show that citrate, cis-aconitate, and isocitrate were all decreased by SDHx mutations reflecting their lowered rates
of production and indicating additional diagnostic utility
to fumarate. Lower levels of fumarate, citrate, cis-aconitate, and isocitrate in SDHx- and VHL-related PPGLs
compared to PPGLs due to RET, NF1, and TMEM127
mutations are consistent with another study demonstrating decreased oxidative phosphorylation in all PPGLs
characterized by a pseudohypoxic signature (30, 33).
Importantly, the present data were generated on massspectrometric instrumentation, which is rapidly becoming
the gold-standard technique for measurements of low molecular weight analytes in the routine diagnostic laboratory. More specifically, the measurements of Krebs cycle
metabolites were performed using an instrument also used
for measurements of plasma and urine metanephrines in
the routine diagnosis of PPGLs (20, 21). Versatility of the
instrumentation, achieved through high sample throughput and ease in changing from one analytical application
to another, ensures utility for multiple applications. The
simplicity of sample preparation also ensures low running
costs of consumables. Furthermore, our finding that succinate, fumarate, and other metabolites are detectable in
paraffin-fixed tissue indicates that the method is not restricted to frozen specimens, but may also be useful for
retrospective analyses of tumor specimens stored by pathology laboratories.
In summary, mass spectrometry-based measurements
of Krebs cycle metabolites in PPGLs provide a useful tool
for identifying underlying SDHx mutations. The method
also offers a window into assessing functionality of mutations; this not only has potential for stratifying risk for
disease aggressiveness, but is also useful to assess functionality associated with gene variants of uncertain significance, thereby enabling damaging mutations to be distinguished from nonfunctional polymorphisms. Such a
case has been described recently in a patient with PPGL,
who carried a novel SDHD missense mutation subsequently identified as nonpathogenic (34). Furthermore,
the method may be applicable to other neoplasms, such as
GI stromal tumors and renal cell carcinomas that may
occur due to mutations of SDHx or genes encoding other
Krebs cycle enzymes (29, 35).
Acknowledgments
Spanish specimens were collected by hospitals through the Spanish National Tumor Bank Network (CNIO).
J Clin Endocrinol Metab, October 2014, 99(10):3903–3911
Address all correspondence and requests for reprints to:
Susan Richter, PhD, Division of Clinical Neurochemistry, Institute of Clinical Chemistry and Laboratory Medicine, Dresden
University of Technology, Fetscherstrasse 74, 01307 Dresden,
Germany. E-mail: [email protected].
This work was funded by the European Union Seventh
Framework Programme (FP7/2007-2013) under Grant Agreement No. 259735 (project ENS@T-Cancer) (S.R., M.P., N.Q.,
G.E., A.A.dC., M.R., E.R., M.M., J.U.R., H.J.T., F.B.), the
Deutsche Forschungsgesellschaft (EI855/1-1) (S.R., M.P., N.Q.,
G.E., J.W.L.), the Fondo de Investigaciones Sanitarias (Project
PI11/01359)(A.A.dC, M.R.), and the Eunice Kennedy Shriver
National Institute of Child Health and Human Development
(K.P.).
Disclosure Summary: The authors have nothing to disclose.
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