The phenotype of SDHB germline mutation carriers: a nationwide study

Introduction

Paragangliomas (PGLs) are rare vascular, neuroendocrine tumors of paraganglia. They derive from either sympathetic chromaffin tissue of the adrenal medulla (also termed pheochromocytoma (PCC)) and extra-adrenal locations (also termed sympathetic PGL (sPGL)) or from parasympathetic tissue of the head and neck (HNPGL) (1). PGLs can occur spontaneously or as part of a hereditary syndrome. Most familial cases of PCC and/or PGL and 10–20% of sporadic cases carry germline mutations. In the Netherlands, succinate dehydrogenase (SDH) germline mutations are responsible for most hereditary cases. The SDHA, SDHB, SDHC and SDHD genes encode for the four subunits of succinate dehydrogenase (also mitochondrial complex II), a key respiratory enzyme that links the Krebs cycle and the electron transport chain (2). The SDHAF2 gene encodes SDH complex assembly factor 2 (SDHAF2), essential for flavination of the SDHA protein and SDH enzyme activity (3). These various germline mutations have distinct phenotypic effects. SDHD-related PGL/PCCs are usually characterized by multiple PGLs, predominantly located in the head and neck region with a low frequency of malignancy. In contrast, SDHB-related disease is often diagnosed as a single tumor (4). Furthermore, SDHB mutation carriers more frequently develop sPGLs, PCCs and metastatic disease than mutation carriers in the other subunits of the SDH gene (5, 6, 7). Although initial malignancy rates as high as 31–97% were reported for SDHB-related PGL (5, 6, 7, 8, 9), we recently reported risks of metastatic disease in SDHB mutation carriers that were considerably lower. A systematic review and meta-analysis reported by Van Hulsteijn et al. demonstrated that the pooled prevalence of metastatic disease was 13% in populations including both asymptomatic SDHB mutation carriers and mutation carriers with manifest PGL, and 23% in studies that included only mutation carriers with manifest disease (10).

SDH mutations have also been linked to non-paraganglionic tumors. In a recent study we strengthened the etiological association of SDH genes with pituitary neoplasia, renal tumorigenesis and gastric gastrointestinal stromal tumors. We also found that pancreatic neuroendocrine tumors may be part of the SDH-related tumor spectrum (11).

Two founder mutations in SDHB have been identified in Dutch PGL families, the c.423 + 1G > A splice site mutation and the c.201-4429_287-933del, p.(Cys68fs) mutation, also annotated as a deletion of exon 3 (12, 13). The aim of this study was to obtain a better impression of the phenotype of SDHB mutation carriers, especially of the two founder mutations. Therefore, we investigated the clinical and biochemical characteristics of disease-affected and unaffected SDHB germline mutation carriers in a nationwide study in seven academic centers in the Netherlands.

Subjects and methods

In this retrospective nationwide study, all SDHB germline mutation carriers diagnosed before 2014 were included in the analysis. All included persons gave written informed consent and in case of persons under 18 years of age, written informed consent was obtained from their parents. Follow-up ended on July 1, 2014 or, when lost to follow-up, the date of the last contact with the endocrinologist or otolaryngologist/head and neck surgeon. We evaluated the genetic, clinical, radiological and biochemical data of SDHB mutation carriers identified in seven of the eight clinical genetics centers of the Netherlands: Leiden University Medical Center (Leiden), University Medical Center Groningen (Groningen), Radboud University Medical Center (Nijmegen), VU University Medical Center (Amsterdam), Erasmus Medical Center (Rotterdam), Academic Medical Center (Amsterdam) and University Medical Center Utrecht (Utrecht). Maastricht University Medical Center was not able to participate for technical reasons. However, they only had identified one germline SDHB mutation carrier. Data from 47 SDHB mutation carriers from the Leiden University Medical Center are previously described by van Hulsteijn et al. (14).

In the academic centers, genetic counseling and DNA testing for mutations in the SDH genes are offered to patients with PCC/sPGL and a positive family history for HNPGL or PCC/sPGL, patients with an isolated PCC/sPGL at an early age (younger than 50 years), and all patients with an HNPGL. If a mutation in the SDHB gene is identified, at-risk family members of the index patients are subsequently invited for genetic counseling and DNA testing for the family-specific SDHB mutation. Screening for germline SDHB mutations is performed by direct sequencing using the Sanger method on an ABI 377 Genetic Analyser (Applied Biosystems) and by multiplex ligation-dependent probe amplification (MLPA) using the P226 MLPA kit (MRC Holland, Amsterdam, the Netherlands). SDHB germline variants are classified as in the international guidelines by Plon et al. (15). In this manuscript we report pathogenic or likely pathogenic variants, including missense mutations in highly conserved regions that are likely pathogenic, as germline mutations.

All SDHB germline mutation carriers were investigated according to structured protocols used for standard care in the Netherlands for patients with a PGL (www.oncoline.nl/familiair-paraganglioom). They were offered annual clinical surveillance for PGL at the departments of otorhinolaryngology and endocrinology. For mutation carriers older than 18 years of age, screening consisted of magnetic resonance imaging (MRI) of the head and neck region once every three years, and MRI or computed tomography (CT) scans of thorax, abdomen and pelvis once every two years. Annual biochemical screening included the measurement of (nor)epinephrine, vanillylmandelic acid (VMA), dopamine, (nor)metanephrine and/or 3-methoxytyramine (3-MT) in two 24-h urinary samples (depending on the academic center which urinary measurement(s) were done), and/or plasma free (nor)metanephrine. In case of excessive catecholamine secretion (i.e. any value above the upper reference limit), radiological assessment by MRI or CT scans of thorax, abdomen and pelvis and/or ¹²³I metaiodobenzylguanidine (MIBG)-scans/positron emission tomography (PET) with 2-deoxy-2-[fluorine-18]fluoro-d-glucose (¹⁸F-FDG PET)-scans/¹⁸F-l-dihydroxyphenylalanine (¹⁸F-DOPA) PET-scans were performed to identify potential sources of excessive catecholamine production outside the head and neck region. In cases without available tumor histology, tumors were classified as paraganglionic based on their specific characteristics on CT and/or MRI. When in doubt, additional nuclear medicine imaging studies were performed in order to confirm the diagnosis.

At the time of this study, there were no national, structured protocols for surveillance in SDHB mutation carriers younger than 18 years of age. Therefore, the method and interval of surveillance in this age category varied between centers. In case of a diagnosis of sPGL, PCC or HNPGL, treatment or intensified periodic examination was offered, guided by the clinical course. In general, for a PCC or sPGL an operation was the preferred treatment of choice. In case of an HNPGL, treatment was guided by the clinical symptoms, tumor characteristics and patient characteristics. Wait and scan policy, radiotherapy or resection were possible treatment options.

An unaffected mutation carrier was defined as a germline mutation carrier without evidence of disease (i.e. HNPGL, sPGL and/or PCC). A disease-affected mutation carrier was defined as a germline mutation carrier with disease, i.e. HNPGL, sPGL and/or PCC.

Malignant disease was defined as the presence of metastases, that is, the presence of chromaffin tissue in locoregional lymphnodes or in non-chromaffin organs distant from the primary tumor, because there are no histological features of the primary tumor that reliably distinguish benign from malignant PGLs.

The study was approved by the Medical Ethics Committee of the Leiden University Medical Center (LUMC; number P13.161), participating centers complied with their local medical ethics committee requirements.

Results

A total of 194 SDHB germline mutation carriers were included: 61 from the Leiden University Medical Center (Leiden), 61 from the University Medical Center Groningen (Groningen), 29 from the Radboud University Medical Center (Nijmegen), 17 from the VU Univer­sity Medical Center (Amsterdam), 18 from the Erasmus Medical Center (Rotterdam), four from the Academic Medical Center (Amsterdam) and four from the Univer­sity Medical Center Utrecht (Utrecht).

In total, 83 men (42.8%) and 111 women (57.2%) were included. The median duration of the follow-up was 2.6 years (range: 0–36). Eleven persons (5.7%) were lost to follow-up: six for unknown reasons, three chose not to pursue any follow-up, one emigrated and one continued the follow-up in a non-participating hospital. Seven persons (3.6%) died: three because of intercurrent disease (lung cancer, metastasized breast cancer and myocardial infarction), one due to progressive disease of a malignant HNPGL (jugular body tumor) with bone metastases, and three due to progressive disease due to a malignant sPGL.

In total, our cohort consisted of 83 (42.3%) disease-affected mutation carriers and 111 (57.2%) unaffected mutation carriers. From the 111 unaffected mutation carriers, 104 have had complete radiological screening (CT/MRI of the head and neck region and CT/MRI of the thorax/abdomen/pelvis). Seven have had either a CT/MRI of the head and neck region (two mutation carriers) or a CT/MRI of the thorax/abdomen/pelvis (five mutation carriers). From the 83 disease-affected mutation carriers, 74 have had complete radiological screening. Nine mutation carriers have had either a CT/MRI of the head and neck region (two mutation carriers) or a CT/MRI of the thorax/abdomen/pelvis (seven mutation carriers). However, all the mutation carriers, who did not have had complete radiological screening by CT/MRI, did had another (total body) imaging study (i.e. ¹²³I MIBG-scans/¹⁸F-FDG PET-scans/¹⁸F-DOPA PET-scans).

There were 65 index patients and 129 relatives of index patients. Of the 129 relatives, 109 persons (84.5%) were unaffected mutation carriers. Four index patients were not affected with HNPGL, PCC or sPGL because these patients had DNA testing for other reasons (one with multiple congenital anomalies, one with two renal cell carcinomas (RCCs) and a gastric gastrointestinal stromal tumor (GIST), one was thought to have an HNPGL, but during radiological follow-up the diagnosis of HNPGL was reversed to no evidence of a tumor and the fourth patient was thought to have a PCC, but this turned out to be a non-functioning adrenal adenoma).

Clinical features

The mean age at first evaluation at the outpatient clinic was 44.8 ± 16.0 years (range 11–76). In total, our cohort comprised of 65 (33.5%) index patients and 129 (66.5%) of their relatives.

Clinical characteristics at the end of follow-up of the cohort as a whole and for four most prevalent Dutch SDHB mutations (deletion exon 3, c.423 + 1G > A, c.654G > A and c.653 G > C) are outlined in Table 2.

Table 2

Clinical phenotypes of specific SDHB germline mutations.

	Total cohort (n = 194)	Exon 3 deletion (n = 60)	c.423 + 1G > A (n = 46)	c.654G > A (n = 19)	c.653G > C (n = 11)
Gender
Man	83 (42.8%)	29 (48.3%)	18 (39.1%)	8 (42.1%)	2 (18.2%)
Woman	111 (57.2%)	31 (51.7%)	28 (60.9%)	11 (57.9%)	9 (81.8%)
Age (mean ± s.d.)a	44.8 ± 16.0	43.2 ± 15.3	51.0 ± 14.5	44.0 ± 18.1	49.1 ± 11.7
Family history positive	129 (66.5%)	40 (66.7%)	35 (76.1%)	18 (94.7%)	8 (72.7%)
HNPGL	54 (27.8%)	18 (30.0%)	11 (23.9%)	1 (5.3%)	3 (27.3%)
1 HNPGL	47	15	10	1	3
2 HNPGL	6	2	1	0	0
3 HNPGL	1	1	0	0	0
CBT	22 (11.3%)	6 (10.0%)	3 (6.5%)	1	2 (18.2%)
Left	11	3	3	0	1
Right	9	4	0	1	1
Bilateral	2	0	0	0	0
VBT	12 (6.2%)	4 (6.6%)	3 (6.5%)	0	1 (9.1%)
Left	6	2	0		1
Right	6	2	3		0
Bilateral	0	0	0		0
JBT	14 (7.2%)	7 (11.7%)	5 (10.9%)	0	0
Left	8	5	3
Right	5	1	2
Bilateral	1	1	0
Tymp	10 (5.2%)	4 (6.7%)	1 (2.2%)	0	0
Left	5	1	1
Right	5	3	0
Bilateral	0	0	0
Other (HNPGL)	1 (right tonsil)	0	0	0	0
Age HNPGLb	45.9 ± 14.2	47.0 ± 14.8	50.6 ± 11.2	27.2	44.8 ± 14.3
Operation HNPGL	27 (50.0%)	8 (44.4%)	4 (36.4%)	0	1 (33.3%)
Radiotherapy HNPGL	15 (27.8%)	8 (44.4%)	4 (36.4%)	0	0
PCC	4 (2.1%)	1 (1.7%)	0	0	1 (9.1%)
Left	3	1			1
Right	1	0
sPGLc	26 (13.4%)	8 (13.3%)	5 (10.9%)	1 (5.3%)	1 (9.1%)
Operation sPGL	25	8 (100%)	5 (100%)		1 (100%)
Malignant PGL/PCC	15 (7.7%)	5 (8.3%)	1 (2.2%)	1 (5.3%)	1 (9.1%)
Other tumorsd	17 (8.8%)	5 (8.3%)	7 (15.2%)g	0	0
Mamma ca.	1	0	1
Renal cell ca.	3e	2	1
Basal cell ca.	2	0	1
Melanoma	2	1	1
Lung ca.	1	0	1
Prostate ca.	1	0	0
Colon ca.	2	0	2
Meibomian gland	1	0	0
Synovial sarcoma	1	1	0
Ovarian ca.	1	0	1
Gastric GIST	2f	0	1
Micro-PRL	1	0	0
Pituitary incidentaloma	1	1	0
Disease status at last follow-up
NED	133 (68.6%)	42 (70.0%)	32 (69.6%)	16 (84.2%)	8 (72.7%)
AWD	43 (22.2%)	13 (21.7%)	9 (19.6%)	1 (5.3%)	3 (27.3%)
LTF	11 (5.7%)	3 (5.0%)	2 (4.3%)	1 (5.3%)	0
DOD	4 (2.1%)	2 (3.3%)	1 (2.2%)	1 (5.3%)	0
DID	3 (1.5%)	0	2 (4.3%)	0	0

^aMean age at presentation at the outpatient clinic in an academic hospital; ^bage at diagnosis HNPGL; ^ctotal cohort: 26 patients with 1 or more sPGLs. Of these 26 patients, five patients had 2 sPGLs; ^dnumber of patients (some patients developed multiple tumors); ^ethere was one patient with two foci of renal cell carcinoma (RCC) on the left side and one RCC on the right side. The other 2 patients both had 1 foci of a RCC; ^fone patient developed three renal cell carcinomas (2 foci on the left side en one on the right side) as well as a gastrointestinal stromal tumor (GIST); ^gone patient with rectal cancer and ovarian cancer, one patient with three RCC as well as a GIST.

AWD, alive with disease; ca., carcinoma; CBT, carotid body tumor; DID, dead of intercurrent disease; DOD, dead of disease; GIST, gastrointestinal stromal tumor; HNPGL, head and neck paraganglioma; JBT, jugular body tumor; LTF, loss to follow-up; NED, no evidence of disease; PCC, pheochromocytoma; PRL, prolactinoma; sPGL, sympathetic paraganglioma; Tymp, tympanicum body tumor; VBT, vagal body tumor.

Of the whole cohort, 54 mutation carriers (27.8%) were clinically affected with one or multiple HNPGLs. Mean age of diagnosis of HNPGL was 45.9 ± 14.2 years (range: 11–77). Carotid body tumors were the most prevalent HNPGLs (in 11.3%), followed by jugular body tumors (in 7.2%) and vagal body tumors (in 6.2%). Twenty-seven carriers (50.0%) had an operation for their HNPGL and 15 (27.8%) received radiotherapy.

Four patients (2.1%) were clinically affected with a PCC. Mean age of diagnosis of PCC was 36.2 ± 16.3 years (range 19–56). Clinical characteristics are detailed in Table 3.

Table 3

Clinical characteristics of the 4 patients with a pheochromocytoma.

Case	Sex	SDHB mutation	Location	Presenting symptoms	Agea	Biochemical phenotype (urinary measurements)	Biochemical phenotype (blood)	Outcome
1	M	Exon 2 deletion	Right	Hypertension, flushes, palpitations	40	NMN elevated, M normal	NA	NED
2	F	c.343C > T	Left	Collaps	28	NA	NA	NED
3	F	Exon 3 deletion	Left	None, brother with SDHB mutation	56	M, NMN, 3-MT slightly elevated	NA	NED
4	F	c.653G > C	Left	Hypertension, flushes	19	NAV	NAV	AWD (vagal body tumor)

^aAge at diagnosis of pheochromocytoma.

F, female; M, male; MN, metanephrine; NA, not assessed; NAV, not available; NED, no evidence of disease; NMN, normetanephrine.

Twenty-six mutation carriers (13.4%) were clinically affected with one or more sPGLs. Mean age of diagnosis of sPGL was 33.4 ± 12.7 years (range: 10–66). None of the 26 mutation carriers suffered from an HNPGL. More than half of the patients with an sPGL had elevated hormone levels. Five carriers had two sPGLs. The sPGLs were mainly located in the abdominal/pelvic region (28 tumors); there were only three thoracic PGLs. Eight persons carried the exon 3 deletion, five the c.423 + 1G > A mutation, two the c.343C > T mutation and another two the c.200 + 1G > A mutation. Twelve of the 26 carriers with one or more sPGLs had metastatic disease and three of them died due to progressive metastatic disease. Clinical characteristics and biochemical phenotypes are detailed in Table 4.

Table 4

Characteristics of 26 patients with sympathetic paragangliomas.

Case	Sex	SDHB mutation	Location sPGL	Agea (year)	Malignant disease	Tumor reduction therapy	Outcome
1	F	c.343C > T	Retroperitoneal and presacral	31	No	Surgery	No evidence of disease
2	F	Exon 3 deletion	Para-aortic	41	No	Surgery (non-radical)	Alive with disease
3	M	c.200 + 1G > A	Retroperitoneal (pararenal)	42	Yes	Surgery, 131I-MIBG therapy, radiotherapy	Alive at age 52, with disease
4	M	Exon 3 deletion	Retropancreatic	11	No	Surgery	No evidence of disease
5	M	Exon 3 deletion	Thoracic (vertebra Th6) and intra-abdominal	10 and 32	Yes	Surgery, chemotherapy radiotherapy 131I-MIBG therapy, RFA	Alive at age 37, without evidence of disease
6	F	Exon 1 deletion	Renal hilum	28	No	Surgery	No evidence of disease
7	M	Exon 3 deletion	Para-aortic abdominal	42	No	Surgery	No evidence of disease
8	F	c.423 + 1G > A	Retroperitoneal	36	No	Surgery	No evidence of disease
9	F	c.725G > A	Para-adrenal	40	Yes	Surgery, Lutetium octreotate therapy	Alive at age 51, with disease
10	F	c.423 + 1G > A	Para-iliac (2 lesions)	19	No	Surgery	No evidence of disease
11	M	c.423 + 1G > A	Para-aortic abdominal	31	No	Surgery	No evidence of disease
12	F	c.653G > C	Retroperitoneal	66	Yes	Surgery, 131I-MIBG therapy	Alive at age 78, with disease
13	M	Exon 3 deletion	Retroperitoneal	37	Yes	Surgery	Alive at age 40, with disease
14	M	Exon 3 deletion	Bladder and retroperitoneal	27	No	Surgery	No evidence of disease
15	M	c.423 + 1G > A	Para-aortic abdominal	38	No	Surgery	No evidence of disease
16	M	c.325A > C	Para-aortic abdominal	30	Yes	Surgery, 131I-MIBG therapy	Alive at age 46, with disease
17	M	c.200 + 1G > A	Bladder	45	Yes	Surgery, radiotherapy, chemotherapy (CVD)	Alive at age 47, with disease
18	M	c.574T > C	Liver hilum	24	No	Surgery	No evidence of disease
19	F	c.727T > A	Retroperitoneal (para-aortic)	52	Yes	Surgery, radiotherapy	Died at age 63, due to intercurrent disease
20	F	c.343C > T	Thoracic	14	No	Surgery	Loss to follow-up
21	F	c.686_725del	Para-aortic abdominal and para-vertebral (Th3/Th4)	39	No	Follow-up	Alive with disease
22	M	c.626C > T	Bladder	42	Yes	Radiotherapy, Firstmappp trial (started June 2014)	Alive at age 52, with disease
23	F	c.423 + 1G > A	Pararenal	31	No	Surgery	No evidence of disease
24	M	Exon 3 deletion	Presacral	28	Yes	Surgery, 131I-MIBG therapy, radiotherapy	Dead of disease: died at age 32 due to progressive disease
25	F	c.654G > A	Bladder	19	Yes	Surgery (primary bladder PGL) sunitinib (metastases)	Dead of disease: died at age 62 due to progressive disease
26	F	Exon 3 deletion	Para-vertebral abdominal	33	Yes	Surgery, 131I-MIBG therapy, radiotherapy	Dead of disease: died at age 37 due to progressive disease

^aAge at diagnosis of sympathetic paraganglioma; *catecholamine measurements at time of primary tumor not available; **catecholamine excess developed with lymph node metastases, not at time of primary tumor; ***catecholamine excess developed at time of malignant disease, not at time of primary tumor.

3-MT, 3-methoxytyramine; CVD, cyclophosphamide, vincristine, dacarbazine; D, dopamine; E, epinephrine; F, female; Firstmappp, randomized, double-blind, phase II, international, multicenter study which is dedicated to determine the efficacy of sunitinib on the progression-free survival at 12 months in patients with progressive malignant pheochromocytoma and paraganglioma; HNPGL, head and neck paraganglioma; M, male; MN, metanephrine; NA, not assessed; NE, norepinephrine; NMN, normetanephrine; PGL, paraganglioma; RFA, radiofrequency ablation; VMA, vanillylmandelic acid.

Out of the whole cohort of SDHB germline mutation carriers, 15/194 (7.7%) developed metastatic PGL. Clinical characteristics, treatment and outcome of the patients with metastatic disease are displayed in detail in Table 5. Treatment of the primary tumor existed of surgery in all patients. None of the 47 mutation carriers described previously have developed metastatic disease since our publication in 2014 (14).

Table 5

Clinical characteristics of patients with metastatic paragangliomas.

Case	Sex	SDHB mutation	Location PGL	Agea (years)	Ageb (years)	Location metastases	Treatment malignant disease	Outcome
1	M	c.200+1G > A	Retroperitoneal (pararenal)	42	45	Bone	Surgery, 131I-MIBG therapy, radiotherapy	Alive at age 52, with disease
2	M	Exon 3 deletion	Thoracic (vertebra Th6)	10	13	Intra-thoracic	Surgery, chemotherapy radiotherapy 131I-MIBG therapy, RFA	Alive at age 37, without evidence of disease
3	F	c.418G > T	Right tonsil	18	20	Lymph nodes, bone (vertebra)	Surgery, radiotherapy	LTF, follow-up till age 22, alive with disease
4	F	c.725G > A	Para-adrenal	40	45	Lymph nodes, bone	Surgery and 177Lutetium octreotate therapy	Alive at age 51, with disease
5	M	c.423+1G > A	Jugular body	48	57	Bone (vertebra)	None	Died at age 57, due to rapidly progressive malignant disease
6	F	Exon 3 deletion	Carotid body	35	66	Lymph nodes, bone	None (not within study period)	Alive at age 66, with disease
7	F	c.653G > C	Retroperitoneal	66	70	Lymph nodes, bone	131I-MIBG therapy	Alive at age 78, with disease
8	M	Exon 3 deletion	Retroperitoneal	37	38	Lymph nodes	Surgery	Alive at age 40, with disease
9	M	c.325A > C	Para-aortic abdominal	30	39	Lymph nodes, bone, lung	131I-MIBG therapy	Alive at age 46, with disease
10	M	c.200+1G > A	Bladder	45	45	Lymph nodes, bone, lung	Surgery, radiotherapy, chemotherapy (CVD)	Alive at age 47, with disease
11	F	c.727T > A	Retroperitoneal (para-aortic)	52	55	Bone	Radiotherapy	Died at age 63, due to intercurrent disease
12	M	c.626C > T	Bladder	42	46	Lymph nodes, bone	Radiotherapy, Firstmappp trial (started June 2014)	Alive at age 52, with disease
13	M	Exon 3 deletion	Presacral	28	28	Bone	Surgery, 131I-MIBG therapy, radiotherapy	Died at age 32 due to progressive disease
14	F	c.654G > A	Bladder	19	58	Lymph nodes, bone	sunitinib	Died at age 62 due to progressive disease
15	F	Exon 3 deletion	Para-vertebral abdominal	33	33	Lymph nodes, bone	Surgery, 131I-MIBG therapy, radiotherapy	Died at age 37 due to progressive disease

^a Age at diagnosis of paraganglioma; ^bage at diagnosis of malignant disease.

CBT, carotid body tumor; CVD, cyclophosphamide, vincristine, dacarbazine; F, female; Firstmappp, randomized, double-blind, phase II, international, multicenter study which is dedicated to determine the efficacy of sunitinib on the progression-free survival at 12 months in patients with progressive malignant pheochromocytoma and paraganglioma; HNPGL, head and neck PGL; LTF, loss to follow-up; M, male; PGL, paraganglioma; RFA, radiofrequency ablation; Th6, 6th thoracic vertebra.

Seventeen mutation carriers (8.8%) developed a total of 21 non-paraganglionic tumors. Three patients developed a total of five (histology confirmed) renal tumors. Four of those tumors were described previously and classified as SDH-deficient renal carcinomas (11, 16, 17). Two patients developed a RCC on one side (one clear cell carcinoma and one SDH-deficient carcinoma), and one patient developed two foci of a RCC on the left side and one on the right side (all three SDH-deficient renal carcinomas). This latter patient also developed an SDH-deficient gastric GIST and has been described previously (11). There was one other patient with an SDH-deficient gastric GIST. Furthermore, there were two patients with a basal cell carcinoma, two with a melanoma, one with a squamous cell lung carcinoma, one with (metastasized) breast cancer, one with prostate cancer, one with a meibomian gland (adeno) carcinoma and one with a (metastasized) synovial sarcoma. In addition, two patients had a rectal cancer and one had ovarian cancer (granulosa cell tumor).

Besides these malignancies, one person developed a microprolactinoma and one person had a non-functioning pituitary incidentaloma, both of which underwent radiological follow-up without available biopsy or surgically-resected material.

Of these 17 mutation carriers with non-paraganglionic tumors, only three patients had also paraganglionic tumors (all three patients had an HNPGL).

The clinical characteristics of the index patients vs relatives are outlined in Table 6 and the age-related disease risk for index patients (probands) vs relatives is outlined in Fig. 1.

Comparison of age at onset in SDHB mutations carriers: index patients (probands) vs relatives. PGL/PCC, risk of (all) paragangliomas/pheochromocytoma; HNPGL, risk of head and neck paraganglioma; sPGL/PCC, risk of sympathetic paraganglioma/pheochromocytoma; malignancy, risk of malignancy.

Citation: European Journal of Endocrinology 177, 2; 10.1530/EJE-17-0074

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Comparison of age at onset in SDHB mutations carriers: index patients (probands) vs relatives. PGL/PCC, risk of (all) paragangliomas/pheochromocytoma; HNPGL, risk of head and neck paraganglioma; sPGL/PCC, risk of sympathetic paraganglioma/pheochromocytoma; malignancy, risk of malignancy.

Citation: European Journal of Endocrinology 177, 2; 10.1530/EJE-17-0074

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Comparison of age at onset in SDHB mutations carriers: index patients (probands) vs relatives. PGL/PCC, risk of (all) paragangliomas/pheochromocytoma; HNPGL, risk of head and neck paraganglioma; sPGL/PCC, risk of sympathetic paraganglioma/pheochromocytoma; malignancy, risk of malignancy.

Citation: European Journal of Endocrinology 177, 2; 10.1530/EJE-17-0074

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Table 6

Clinical characteristics of index patients and relatives.

	Age (mean ± s.d.)	Follow-up (median, year)	HNPGL (%)	PCC (%)	sPGL (%)	Malignant PGL/PCC
Index patients (65)	43.6 ± 14.8	4.5	38 (58.5)	3 (4.6)	21 (32.3)	15 (23.1)
Relatives (129)	45.4 ± 16.6	2.0	16 (12.4)	1 (0.8)	5 (3.9)	0

To explore potential differences in clinical phenotypes related to the specific mutations within the SDHB gene, carriers of the two most common SDHB mutations in the Netherlands (exon 3 deletion and c.423 + 1G > A) were compared. Statistical analyses showed no significant differences in number and location of HNPGLs, sPGLs or PCCs, nor in the occurrence of malignant disease or other tumors.

Discussion

In this nationwide multicenter study we assessed the phenotypes of 194 SDHB germline mutation carriers. Our cohort consisted of 83 (42.8%) disease-affected mutation carriers and 111 (57.2%) unaffected mutation carriers. Fifty-four carriers (27.8%) were clinically affected with one or multiple HNPGLs. Only four patients (2.1%) were clinically affected with a PCC and 26 (13.4%) with one or more sPGLs. Fifteen patients (7.7%) developed metastatic disease.

Previous studies have reported much higher rates for developing PCC and sPGLs, 18–52% and 59–84% respectively (5, 6, 8, 18). For various reasons, it is quite difficult to directly compare our results with those reported in the literature. The majority of previously published studies include a high proportion of index patients. This may result in ascertainment bias and therefore overestimation of the risk of developing HNPGL, PCC, sPGL or malignant disease. A recently published study by the French network on PGL/PCC in SDHx mutation carriers included 124 SDHB mutation carriers, 39 (31%) of whom were index patients and 85 persons (69%) were relatives of index patients (19). This cohort seems to resemble the proportions of our study cohort, and the prevalences of PCC (1.6%) and sPGL (6.5%) found in their study are more comparable to the results in our current study (2.1% and 13.4% respectively). The low percentages of PCC/sPGLs reported in France and in the present study indicate that the high percentages described in several other studies are likely to be the result of ascertainment bias. Furthermore, it should be noted that the percentages mentioned in most studies are calculated using the total number of tumors divided by the total number of patients with any tumor, thereby taking only disease-affected persons into account. Removal of all unaffected mutation carriers from our cohort (111 subjects) would give a figure for PCC of 4 in 83 (4.8%) and 26 in 83 (31.3%) for sPGL. Even if we take only disease-affected individuals into account, our figures are substantially lower than in previous studies that have assessed clinical characteristics in SDHB mutation carriers. By contrast, we found a relatively high frequency of HNPGLs (27.8%) among SDHB mutation carriers compared with other studies (3–31%) (5, 6, 8, 18) and even when compared with that of the French network (14.5%) (19). If only the disease-affected mutation carriers were taken into account, the prevalence of HNPGL was as high as 54/83 (65.1%) in our cohort, nearly double the frequency reported previously in disease-affected subjects (5, 6, 8). This might in part be explained by the observation that in our study the proportion of HNPGL patients with a positive family history (i.e. non-index HNPGL patients) is 29.6% (16/54). The large majority of these patients had no symptoms and had not yet come to medical attention. The genetic testing of relatives and structured follow-up protocols of persons with a SDHB mutation in the Netherlands identifies a relatively high number of asymptomatic mutation carriers, with or without tumors, allowing for a more accurate representation of the phenotype of SDHB mutation carriers.

The observation that the majority of SDHB-linked patients develop an HNPGL furthermore underlines the importance of radiological screening of the head and neck region in SDHB mutation carriers.

Only fifteen patients (7.7%) in the entire cohort, including both disease-affected and unaffected mutation carriers, developed metastatic PGL. In three of these patients (20%) the primary tumor was an HNPGL (including one in the tonsil) and in 12 patients (80%) the primary tumor was an sPGL. Removal of all unaffected mutation carriers (111 subjects) results in a prevalence of metastatic disease by 18.1% (15/83) in PGL/PCC patients. Taking into account only the sPGL patients, the malignancy risk is as high a 46.2% (12/26). For HNPGL patients, this malignancy rate was 5.6% (3/54). This means that the malignancy risk for patients already suffering from an sPGL is high, which has implications for the follow-up of those patients. Srirangalingam et al. reported metastatic PGL in five of 16 (31%) disease-affected subjects (8). However, the malignancy rate for the entire cohort was 16% (5/32). The rates of malignancy reported in the literature are calculated based on disease-affected subjects and vary from 31 to 97% (5, 6, 7, 8, 9). These reported malignancy rates are however most likely also inflated because of selection bias in referral-based studies. Alternatively, the discrepancy in malignancy rates may also be a result of variable follow-up times (7, 8). A recent systematic review of prevalence studies comprising both asymptomatic SDHB mutation carriers and SDHB mutation carriers with manifest non-malignant PGL documented a pooled risk for developing metastatic PGL of 13 and 23% respectively (10), also much lower than previously reported (20, 21). In the fifteen patients with metastatic PGL, we found a wide range of time to metastatic disease (0–39.2 years). This is in line with previously published results. Timmers et al. found a range from 0 to 17 years (7) and Srirangalingam et al. between 1.5 and 25 years (8). Because it is not possible to diagnose malignancy based on histopathology of the primary tumor, only if metastatic disease is present, the current and previously reported wide ranges of time to metastatic transformation underscore the need for an extended follow-up in patients with an SDHB mutation, especially in disease-affected mutation carriers. The median duration of follow-up is 2.6 years in this study, which is a limitation of this study. However, the follow-up time is relatively short due to a shorter follow-up of relatives compared to index patients. Future studies with a longer duration of follow-up are needed to validate our results.

Our findings show a relatively mild phenotype of SDHB mutations in the Netherlands. One might hypothesize that this could be associated with the low altitude and therefore relatively high oxygen levels in the Netherlands (22). However, studying a large cohort from a single country provides a more homogeneous study population and the inclusion of unaffected mutation carriers should provide better information on actual tumor risks than series that include mainly index patients (18). The high proportion of unaffected mutation carriers in our study seems to reflect an active testing protocol in the Netherlands of at-risk family members of the index patients, who are advised to undergo genetic counseling and DNA testing for the family-specific SDHB mutation. Lower lifetime cancer risks have also been established for other genetic tumor syndromes following the inclusion of unaffected mutation carriers, one well-known example being pathogenic BRCA1/2 gene variants (23). Lower cumulative lifetime risks of breast cancer followed from analyses that excluded index patients while including first-degree relatives.

In conclusion, in this nationwide study which allowed for the inclusion of SDHB germline mutation carriers identified in the Netherlands, we found a lower rate of metastatic disease and a relatively high number of HNPGLs compared with previous reports of referral-based cohorts. This is most probably not a regional phenomenon but the result of the more comprehensive inclusion of unaffected mutation carriers, underlining the importance of including both disease-affected and unaffected individuals in studies that assess the phenotype of germline mutations. It furthermore highlights the importance of thorough tumor screening protocols that include radiology of the head and neck region in SDHB mutation carriers.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this study.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Author contribution statement

Nicolasine D Niemeijer helped in study design, data collection, data analysis and preparation of the manuscript. Johannes A Rijken helped in study design, data collection and preparation of the manuscript. Karin Eijkelenkamp, Anouk N A van der Horst-Schrivers and Michiel Kerstens helped in data collection and editing of the manuscript. Carli M J Tops and Anouk van Berkel helped in data collection. Henri J L M Timmers helped in data collection and editing of the manuscript. Henricus P M Kunst, C René Leemans, Peter H Bisschop, Koen M A Dreijerink, Marieke F van Dooren and Jean-Pierre Bayley helped in data collection. Alberto M Pereira helped in data analysis and preparation of the manuscript. Jeroen C Jansen helped in data collection and preparation of the manuscript. Frederik J Hes and Erik F Hensen helped in study design, data collection and preparation of the manuscript. Eleonora P M Corssmit helped in study design, data collection, data analysis and preparation of the manuscript.