Genotype–phenotype associations in PPGLs in 59 patients with variants in SDHX genes

Introduction

Phaeochromocytomas and paragangliomas (PPGLs: PCCs (MIM: 171300), PGL1 (MIM: 168000), PGL2 (MIM: 601650), PGL3 (MIM: 605373), PGL4 (MIM: 115310), PGL5 (MIM: 614165), PGL6 (MIM: 618464)) are tumours of the adrenal medulla and extra-adrenal sympathetic nervous system, respectively, that often secrete catecholamines. Paragangliomas (PGLs) arising from the parasympathetic nervous system are often non-secreting tumours located in the head and neck region (termed head and neck paragangliomas (HNPGLs)) (1). Because of the strong heritability, genetic testing is recommended in all patients with PPGLs and, when a variant is detected, also screening of their family members (1, 2, 3, 4, 5). Markers of poor prognosis in PPGLs have been identified in different studies, including tumour size >5 cm, vascular invasion, SDHB variants and norepinephrine secretion (6). There is, however, little information about the clinical phenotype associated with the individual genetic variants (6, 7).

PPGLs are very heterogeneous tumours and more than 20 predisposing genes have been identified (6). In two-thirds of the PPGL patients, a pathogenic germline or somatic variant can be found and PPGLs have been divided into distinct biological subgroups (kinase signaling, pseudohypoxia and Wnt signaling) on the basis of these variants (6). Most PPGLs are non-metastatic tumours but approximately 15% are malignant (6). Malignancy status is given to metastatic disease at distant sites unrelated to the paraganglia tissues (6). The overall 5-year survival for malignant PPGLs is approximately 60% (8). Germline variants in succinate dehydrogenase (SDH)-A, -AF2, -B, -C, and -D (MIM: 600857; MIM: 613019; MIM: 185470; MIM: 602413; MIM: 602690) have been found to account for approximately half of the hereditary PPGLs (9) and for up to 15% of all patients with PPGLs (10).

The SDHX genes belonging to the SDH gene family encode a protein complex, which is involved in the Krebs cycle and the respiratory chain and are classified as tumour suppressors (9, 10, 11, 12). A germline variant of SDHB is the best-known genetic predictor of malignancy as approximately 50% of tumours progress to metastatic disease (6, 13). Two-thirds of malignant PPGLs, however, do not harbour SDHB variants (6) and other predictive markers of poor prognosis have been investigated. The PASS (phaeochromocytoma (PCC) of the adrenal gland scaled score) was developed to identify those PCCs (not PGLs) that have metastatic potential based on a score ≥4 (14). The PASS method has a number of limitations in the clinical setting as the inter- and intra-observer variability is high (6, 15, 16). A recent study found that the PASS was best used clinically to rule out, rather than rule in, malignancy potential for PPGLs due to low positive predictive value and relatively higher negative predictive value (17).

Thus, existing prognostic markers cannot fully predict PPGL behaviour (6, 18), which is a clinical challenge. To our knowledge, there is no previous systematic study of the genotype–phenotype associations in patients with solely germline variants in SDHX genes. In this study, we aim to characterise the possible associations between genotype on the one hand and the clinical and histopathological presentation on the other in 59 individuals diagnosed with germline variants in SDHX genes in the setting of a single tertiary referral centre.

Methods

Study cohort

The study cohort comprised all individuals diagnosed with a variant of one of the SDHX genes between 1 January 2006 and 1 March 2019 who were followed in the Department of Medical Endocrinology, Copenhagen University Hospital, Rigshospitalet. All individuals with a proven variant in the SDHX genes (class 3, 4, or 5) were included. The persons were identified by their social security number in the Danish National Patient Registry. Genetic screening, variant classification, and clinical workup were carried out at the Centre for Genomic Medicine, Copenhagen University Hospital, Rigshospitalet. The study was approved by the Data Protection Agency (I-Suite number 6712) and the National Committee on Health Research Ethics, Denmark (H-1803546). The approval included a dispensation for collecting an informed consent and the study was performed according to the flow chart in Fig. 1.

View Full Size

Flow chart of the study. CRH, corticotropin-releasing hormone; HNPGL, head and neck paraganglioma; ki-67, antigen associated with cellular proliferation; NSE, neuron-specific enolase; PASS, phaeochromocytoma of the adrenal gland scaled score; PCC, phaeochromocytoma; PGL, paraganglioma; PPGL, phaeochromocytoma and paraganglioma; SDHX, succinate dehydrogenase genetic variants; SS receptor, somatostatin receptor; VIP, vasoactive intestinal peptide.

Citation: Endocrine Connections 9, 8; 10.1530/EC-20-0279

• Download Figure
• Download figure as PowerPoint slide

Results

The characteristics of the cohort are shown in Table 1.

In total, 59 individuals were diagnosed with an SDHX variant during a 13-year period. We were able to retrieve clinical records for 58 of these. One medical file was lost and for this patient we were only able to retrieve the pathology report and genetic test. A total of 24 SDHX variant positive patients underwent surgery (Fig. 1).

The median age at PPGL diagnosis was 42.5 years. There was no sex difference between the median age at tumour diagnosis of women (40.5 years (n = 14)) compared to men (45.5 years (n = 10), (P = 0.62)) or for the number of individuals with an SDHX variant (30 female, 29 male, P = 0.34) (Table 1).

Most patients were diagnosed with variants in the SDHB gene (51/59). Only two, four, and two individuals were positive for variants in SDHA, SDHC, and SDHD, respectively, while no patients were positive for SDHAF2. A total of 18 PPGLs and 5 PCCs were diagnosed. Proof of metastases was found in 12 out of 59 patients. Seventeen out of 51 SDHB variant patients were diagnosed with PPGLs. One of the SDHA variant positive patients (n = 2) was diagnosed with a PGL. All SDHC (n = 4) and one SDHD patient developed PPGLs. Three patients were diagnosed with renal cell carcinoma, two with an SDHB variant and one with a variant of the SDHD gene (Table 1).

The histopathological characteristics of the tumours are shown in Supplementary material (see section on supplementary materials given at the end of this article).

In total, the cohort had 12 probands and 41 family members with a class 4 or 5 variant and 6 individuals with a class 3 variant. Among the 41 family members, two individuals were diagnosed with tumours at the first screening and two with tumours during routine screenings after 2 and 7 years. Three family members with a class 4 or 5 variant that had been treated for tumours earlier in life and were diagnosed with the genetic variant many years later during family screening. Thirty-four family members with a class 4 or 5 variant have been followed for an average time of 48.8 months (range: 11 days to 136 months) without tumour detection.

Comparison of patients with class 4 and 5 variants (n = 19; 1 SDHA, 15 SDHB and 3 SDHC) showed that 10 of 19 developed metastatic disease and that the age at tumour diagnosis was related to the genotype with family B1 presenting with tumours at a lower age than family B3 (Fig. 2). The patients with class 4 and 5 variants in SDHB presented mostly with PGLs; only one patient had both a PCC and a PGL. Three SDHB patients had non-metastatic PGLs and both cases with renal cell carcinomas were confined to the kidney. Within the SDHB variant group, two genetic variants showed a specific age distribution of first tumour diagnosis. In family B1, PGLs were detected between the age of 12 and 28 years and 75% were with metastatic disease. In family B3, metastatic or non-metastatic PGLs or renal cell carcinomas confined to the kidney were diagnosed between the age of 28 and 57 years. Family B4 had the same variant as family B3 and the patients were diagnosed with either renal cell carcinoma confined to the kidney or metastatic PGL at the age of 43 and 50 years.

View Full Size

Age at first tumour diagnosis (PPGL or RCC) in patients with class 4 and 5 variants of SDHA (n = 1), SDHB (n = 15), and SDHC (n = 3) genes (open circles depict non-metastatic tumours, stars show metastatic disease). None of the SDHD variants were class 4 or 5. AM1, SDHA family, male 1; B1F1 to B8F1, SDHB families 1, 3, 4–8; F, female; M, male; CF3 to CM2, SDHC families; RCC, renal call carcinoma; PPGL, phaeochromocytoma and paraganglioma; SDH, succinate dehydrogenase.

Citation: Endocrine Connections 9, 8; 10.1530/EC-20-0279

• Download Figure
• Download figure as PowerPoint slide

All patients with SDHC variants were diagnosed after the age of 50 years. Two patients with metastatic disease were unrelated and older than the patient with the non-metastatic tumour (Fig. 2). In total, six variants were classified as class 3 (three SDHB, one SDHC, and two SDHD).

Discussion

To our knowledge, this is the first systematic study of a single tertiary centre cohort of 59 individuals with germline SDHX variants describing the associations between the genotype on the one hand and both the clinical and histopathological phenotype on the other. SDHB variants were the most frequent, which is in accordance with previous reports (9, 10, 12, 18, 21). Two patients were diagnosed with a variant in SDHA and four patients were diagnosed with SDHC variants, which corroborates previous findings that these are much less common (9, 10, 12, 22). Only two individuals were diagnosed with SDHD variants, although these have been described as more common in other populations (9, 10, 12).

Our patient cohort of SDHX variant positive individuals consisted of almost equal numbers of females (30/59) and males (29/59) which was expected from the autosomal dominant heritance with incomplete penetrance of SDHA, -B, -C, -D or -AF2 variants located at chromosomes 5, 1, 1, 11, and 11, respectively. There was also no significant sex difference in whether or not the patient developed tumours. The SDHA positive family (n = 2) has, to our knowledge, a novel class 4 variant not previously reported. This variant was associated with PGLs already at the age of 19 years, thus underlining the necessity for continuous screening. SDHB variants are common and have a high penetrance compared to other SDHX variants resulting in development of abdominal PGLs, HNPGLs, and SDH deficient renal carcinomas (10, 12, 22). The class 4 and 5 variants in our cohort predisposed to metastatic or non-metastatic PGLs and renal cell carcinomas without local invasion or metastases which correlated with previous findings (9, 10, 12). In family B2, no heterozygous individual with a class 5 missense variant (c.769C>G, p.Leu257Val) in exon 8 of the SDHB gene has as yet developed PPGLs. Literature, however, suggests an increased risk (20), indicating a low penetrance for this specific variant. The homozygous proband’s clinical course supports the international recommendation that sequencing of SDHB, -D, and possibly -C should be performed in patients suspected for or diagnosed with multisystem mitochondrial disorder and complex II deficiency (20).

The patients and healthy family members were included in a follow-up programme according to available international guidelines, from the year 2016 those of European Society of Endocrinology (3) with genetic testing and regular tumour screening (plasma or urinary catecholamines and chromogranin A every year, imaging every 1–2 years). This revealed tumour development before the onset of symptoms at younger ages than the proband (family B1). Tumours were consequently smaller at diagnosis and more often non-metastatic (family B1). Furthermore, one female and one male were diagnosed with PGL at the very first screening (family B3).

Distinct differences were found in the clinical picture and histopathology between various genetic variants which in the future may prove useful for the development of more individualised clinical guidelines for follow-up. Notably, families B1 and B3 showed distinct differences in age of first tumour diagnosis. Families B3 and B4 carry the same start loss variant (class 4 variant in exon 1 of SDHB, c.3G>A, p.Met?) that is likely pathogenic (11) and both families had clinical signs of poor prognosis. Families B5 and B7 support the hypothesis that a Ki-67 index >1% is a possible marker for malignancy in PPGLs (6, 23).

Variants in the SHDC gene usually predispose to non-metastatic and multiple HNPGLs (9, 10, 12). All four of our SDHC variant individuals developed tumours (HNPGLs and PPGLs), two with proof of metastasis and two showed catecholamine-related symptoms, which, according to the literature, are two very rare findings (9). One of the individuals with proof of metastasis had a class 3 variant further underlining that the tumour classification alone cannot predict clinical the phenotype (24). Larger studies of the genotype–phenotype associations are needed to elucidate how the different genetic variants in PPGLs may be stratified into prognostic risk categories. Furthermore, a study suggested that the variant type (truncating vs missense) in SDHD had an important clinical impact (25).

Variants in the SDHD gene usually predispose to multiple HNPGLs and less often PPGLs (10). However, in our cohort, the SDHD positive individuals developed malignant renal cell carcinoma and metastatic phaeochromocytoma. According to the literature, only one previous case of renal cell carcinoma has been reported in a patient with an SDHD variant (26).

Our study was based on the Danish National Patient Registry, which ensures that patient records are easily found. In fact, only one clinical record was missing and we obtained both genetic test results and pathology reports from all relevant individuals. The registry has previously been validated as a valuable tool for research in rare diseases (27). Our study included all patients diagnosed since 1 January 2006 in the Department of Endocrinology, Copenhagen University Hospital, Rigshospitalet, resulting in 59 patients. A national cohort would increase the power to detect differences between specific genotypes and corresponding phenotypes and possible regional differences. It has previously been shown that the distribution of RET variants in Denmark differs from other populations, possibly due to a founder effect (28), and that the same could be the case for SDHX variants, as other countries have shown differences in clinical presentation of SDHX mutations (29).

The clinical records obtained did not always include a description of subjective symptoms. This is a well-known drawback of retrospective studies and a potential source of recall bias or bias due to clinical practice, that is, whether the individual was asked about subjective symptoms or not. Some of the individuals have only recently been diagnosed with a familial genetic variant in SDHX and may not yet have developed tumours because of the short follow-up time or young age. The pathology reports obtained for this study were carried out by different pathologists and, therefore, the interpretation of the findings may be subject to observer variation.

We decided to define malignancy for PPGLs as proof of metastases based on the guidelines (3, 30). Together with the genotype, Ki-67% labelling index, secretion of hormones, tumour size, location, and clinical symptoms, we included components of the PASS score to cover all possible markers of poor prognosis reported in the literature (6). The PASS was not originally developed for PGLs and, therefore, there is some uncertainty in applying the components of the PASS to these tumours. The clinical features and histopathological characteristics are, however, similar for PCCs and PGLs (31).

In summary, our study corroborates previous findings of poor prognostic markers (6, 12). Furthermore, we found that the genetic variants are linked to the clinical and histopathological phenotype and prognosis and therefore useful for the decision of clinical follow-up. Family screening for SDHB variants allowed an earlier detection of smaller tumours with better prognosis in one family (B1) and detection of tumours at the first screening in another (B3). This supports current guidelines recommending family screening. Within our cohort, two SDHB variants (a deletion of exon 4 and 5 and c.3G>A, p.Met1? in exon1) were identified in individuals with specific phenotypes which may be relevant in future counselling. The SDHA, SDHC and SDHD patients in our cohort included malignant phenotypes, underlining the necessity to screen for all SDHX variants and, if positive, we recommend family screening to improve early tumour detection and thereby better prognosis. The development of an objective combined algorithm with clinical, genetic, morphological, and biochemical factors may be the future for clinical individualized risk stratification.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/EC-20-0279.

Declaration of interest

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

Funding

Ulla Feldt-Rasmussen’s research salary was supported by an unrestricted research grant from the Novo Nordisk Foundation.

Author contribution statement

Ulla Feldt-Rasmussen was the supervisor and performed project planning, data analysis and interpretation. Ailsa Maria Main performed data collection, data analysis, statistics, interpretation, and article writing. Åse Krogh Rasmussen was the co-supervisor and performed project planning, data analysis and interpretation. Caroline Maria Rossing performed collection of existing genetic results from database, genetic reruns by NGS, interpretation of genetic results, and provided expert counselling on clinical genetics and formulated the section on genetics under ’methods’. Line Gutte Borgwardt performed collection of existing genetic results from database, genetic reruns by NGS and interpretation of genetic results. Birgitte Grønkær Toft provided expert counselling on pathology reports.