Original contributionImmunohistochemistry for SDHB triages genetic testing of SDHB, SDHC, and SDHD in paraganglioma-pheochromocytoma syndromes
Introduction
Pheochromocytomas and paragangliomas are rare tumors with a combined estimated annual clinical incidence of 3 per million [1]. By convention, tumors arising within the adrenal gland are known as pheochromocytomas, whereas morphologically identical tumors arising elsewhere are termed paragangliomas[2]. Paragangliomas are further divided into sympathetic paragangliomas that predominantly arise from the intraabdominal sympathetic trunk and usually produce catecholamines and parasympathetic paragangliomas that are distributed along the parasympathetic nerves of the head, neck, and mediastinum and are rarely functional [2]. Paraganglioma may also arise outside the normal distribution of sympathetic and parasympathetic ganglia predominantly in the cauda equina and orbit where they are usually not functional [2], [3].
In contrast to the classic maxim that 10% of pheochromocytomas are inherited, it is now estimated that up to 30% of all pheochromocytomas are familial [4], [5], [6]. To date, autosomal dominant germline mutations of 7 genes have been described in association with these tumors [2], [4], [5], [6], [7], [8]. Although surgical pathologists are well aware of MEN2, Von Hippel–Lindau (VHL) syndrome, and NF1 because of the constellation of other classic tumors with which they are associated, there is less awareness of the recently described familial paraganglioma-pheochromocytoma syndromes (PGL1, PGL2, PGL3, and PGL4) associated with germline mutations of SDHD, SDHAF2, SDHC, and SDHB, respectively. PGL1 occurs with mutations of SDHD that maps to 11q23 and disease is only inherited from the paternal line presumably as a result of maternal imprinting [9]. It manifests more commonly with head and neck paragangliomas, has a higher penetrance, and has a greater incidence of multifocality compared with tumors bearing germline SDHB mutations [10], [11]. It is probably not associated with other tumor types [1], [4], [5]. PGL2 is rare and generally manifests as head and neck paraganglioma [2]. The gene responsible for PGL2, initially termed SDH5 but now known as SDHAF2, has only recently been described and maps to 11q13.1 [7]. PGL3 results from mutations of SDHC (1q21-23) and is also rare. To date, only a few kindreds have been described [12]. Affected individuals develop tumors that are unifocal, benign, and usually located at the carotid body [1], [5], [12]. There may also be an increased risk of gastrointestinal stromal tumor [4]. PGL4 occurs with mutations of SDHB 1p36 and more commonly manifests as intraabdominal sympathetic paragangliomas. It has a much higher risk of malignant behavior that may be greater than 50% [1], [5], [13], [14]. There is a also a suggestion of an increased risk of renal cell carcinoma and gastrointestinal stromal tumor [4], [11], [15], [16], [17]. SDH mutations in tumors are virtually always associated with germline mutations, and we are only aware of 2 reports of somatic mutation (1 SDHB and 1 SDHD) in the absence of germline mutation [14], [18], [19].
The familial basis of pheochromocytomas and paragangliomas may not be recognized clinically because of incomplete penetrance, phenotypic heterogeneity, lack of clinical awareness, and, in the case of SDHD-associated disease, parent of origin effect masking family histories [20]. Therefore, germline mutations are detected in between 7% and 24% of subjects with pheochromocytoma clinically considered sporadic [6], [8], [21]. Neurofibromatosis should be clinically apparent because of the classic cutaneous features, but testing for the other genes should always be considered in any individual with pheochromocytoma/paraganglioma even in the absence of syndromic features [4], [13], [22], [23], [24]. Whether complete mutation testing is justified in every instance of apparently sporadic pheochromocytoma or should be limited to those with increased risk such as young age, multiple tumors, malignant behavior, or extraadrenal location is controversial because of the cost of the techniques involved. However, several groups have recommended universal genetic testing in pheochromocytoma and paraganglioma with the order in which the genes are tested being directed by the location of the tumors and the associated clinical features [25], [26]. Such an approach is necessarily associated with significant cost, up to US $2700 for all 3 SDH genes alone and up to US $4100 when combined with RET and VHL gene tests [27].
Recently, van Nederveen and colleagues [6] proposed that negative immunohistochemical staining for SDHB is highly specific for pheochromocytomas and paragangliomas associated with SDHB, SDHC, or SDHD gene mutations. In their series, all 65 (100%) patients with genetically proven VHL and MEN2 had positive SDHB staining. Furthermore, all 102 (100%) patients with genetically proven mutations of SDHB, SDHC, or SDHD displayed negative immunohistochemistry results (IHC).
Our institution is a referral center for genetic testing in pheochromocytoma associated syndromes for Australia and New Zealand. Testing commenced for VHL in 1998, SDHD in 2000, and SDHB in 2001. We sought to test the utility of IHC for SDHB on routine formalin-fixed, paraffin-embedded tumor tissue on our own independent cohort of 58 patients with known mutation status for the SDH genes. To assess the incidence of SDH mutations in a truly unselected cohort for the first time, we then went on to perform IHC for SDHB on all 95 other individuals who received a tissue diagnosis of pheochromocytoma/paraganglioma over a 20-year period. When the 10 individuals who had surgery elsewhere but presented for genetic testing to our services were excluded on the basis that they introduce a selection bias, we were therefore able to estimate the incidence of SDH abnormalities in a truly unselected group of 143 patients representing a cross-section of individuals presenting to both tertiary referral centers and community hospitals. Many of these individuals would have been excluded from other incidence studies because they were seen by physicians who were relatively unfamiliar with these syndromes and were not considered for genetic testing.
Section snippets
Methods
Paraffin-embedded tumor blocks were available from 58 subjects who had undergone genetic tests for pheochromocytoma/paraganglioma associated genes at the Cancer Genetics Unit of the Kolling Institute (Sydney, Australia). Genetic testing had been performed either by polymerase chain reaction and direct sequencing as previously described [28], [29] or by denaturing high-performance liquid chromatography (dHPLC) and confirmatory direct sequencing [26]. Only the 2 NF1 patients had not undergone
Results
The results are summarized in Tables 1 to 3. Both independent pathologists reached the same scoring result for all tumors (κ score = 1.0). Of the 58 cases with known genetic status, 6 patients had SDHB mutation, 5 had SDHD mutation, and 1 had SDHC mutation; and all of these showed either negative or weak diffuse staining. The 6 SDHB mutation-positive cases were completely negative on IHC. Completely negative staining was also seen in the SDHC mutated case but only 1 of the 5 SDHD mutated cases.
Discussion
SDHA, SDHB, SDHC, and SDHD are encoded in the nucleus but assembled in the mitochondria to form the 4 subunits of the mitochondrial complex II (succinate dehydrogenase/succinate-ubiquinone oxireductase) that links the Krebs cycle and the respiratory electron transport chain [2], [30], [31], [32]. SDHA and SDHB (the catalytic components) form the hydrophilic part of the complex that acts in the Krebs cycle, whereas SDHC and SDHD (the anchoring components) are hydrophobic integral membrane
Acknowledgments
The authors wish to acknowledge the assistance of Dr Rachael Smith (Royal Hobart Hospital Pathology), Dr Brian Beer (Northern Rivers Pathology), Dr Adrienne Morey and Dr Wade Barrett (Sydpath Pathology St Vincent's Hospital), Dr Annabelle Mahar (Royal Prince Alfred Hospital Pathology), and Dr Katherine Tucker (Prince of Wales Hospital) who assisted in tracing paraffin blocks to be involved in this study.
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Dr Gill and Dr Benn contributed equally to this work.