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Succinate dehydrogenase (SDH) and mitochondrial driven neoplasia
Pathology (June 2012) 44(4), pp. 285–292
REVIEW
Succinate dehydrogenase (SDH) and mitochondrial driven neoplasia ANTHONY J. GILL*{ *Department of Anatomical Pathology and Northern Cancer Translational Research Unit, Royal North Shore Hospital, St Leonards, and {University of Sydney, Sydney, New South Wales, Australia
Summary The genes for the succinate dehydrogenase (SDH) subunits SDHA, SDHB, SDHC and SDHD are encoded in the autosome. The proteins are assembled in the mitochondria to form the mitochondrial complex 2, a key respiratory enzyme which links the Krebs cycle and the electron transport chain. Thirty percent of phaeochromocytoma and paraganglioma (PHEO/PGL) are hereditary and perhaps as many as half of these familial cases are caused by germline mutations of the SDH subunits. Negative immunohistochemical staining for the SDHB subunit identifies PHEO/PGL associated with germline mutation of any of the mitochondrial complex 2 components and can be used to triage formal genetic testing of all PHEO/PGL for SDH mutations. PHEO/PGL associated with SDHA mutation also show negative staining for SDHA as well as SDHB. A unique subgroup of gastrointestinal stromal tumours (GISTs) are driven by mitochondrial complex 2 dysfunction. These SDH deficient GISTs can also be definitively identified by negative staining for SDHB and show distinct clinical and morphological features including frequent onset in childhood and young adulthood, gastric location, a tendency to multifocality, absence of KIT and PDGFRA mutations, a prognosis not predicted by size and mitotic rate and a tendency to indolent behaviour of metastases. Some of these SDH deficient GISTs are driven by classical SDH mutations, but the precise mechanisms of tumourigenesis in many (including those associated with the Carney triad) remain unknown. Germline SDHB mutation is associated with a newly recognised type of renal carcinoma which commonly but not always demonstrates distinctive morphology and can also be recognised by negative staining for SDHB. Immunohistochemistry for SDHB therefore has emerged as a useful tool to recognise these distinct neoplasias driven by mitochondrial complex 2 dysfunction and to triage formal genetic testing for the associated syndromes. Abbreviations: GIST, gastrointestinal stromal tumour; PHEO/PGL, phaeochromocytoma and paraganglioma; SDH, succinate dehydrogenase. Key words: Gastrointestinal stromal tumour, GIST, paraganglioma, phaeochromocytoma, SDHB. Received 23 February, revised 7 March, accepted 12 March 2012
INTRODUCTION Otto Heinrich Warburg (1883–1970) was awarded the 1931 Nobel prize in medicine or physiology for his groundbreaking work on the nature and mechanisms of action of the respiratory enzymes.1 He is better known now for his demonstration that even in the presence of abundant oxygen most cancer cells Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0b013e3283539932
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produce energy predominantly by glycolysis (resulting in lactate production) rather than by the far more efficient process of mitochondrial oxidative respiration on which most nonneoplastic cells rely. The Warburg effect, as this phenomenon and the subsequent demand for large amounts of glucose by malignant cells has become known, has been a poorly understood but consistently observed occurrence in malignant cells. It is the basis of the routine detection and monitoring of human malignancies by fluorodeoxyglucose positron emission tomography (PET scan) and the development of agents which target the ‘addiction’ of malignant cells to glucose is an area of much promising research in the field of drug development.2 However, Warburg went further than to merely observe the link between deficient mitochondrial oxidative respiration and neoplasia. He steadfastly maintained that defective respiration caused malignancies. As he wrote in 1956: ‘there is only one common cause into which all other causes of cancer merge, the irreversible injuring of respiration.’3 Warburg’s belief that altered mitochondrial respiration is the primary cause of cancer (the Warburg hypothesis) has been surpassed by the understanding that malignancies are caused primarily by DNA mutations which lead to growth dysregulation and genomic instability. Mitochondrial dysfunction appears to be a secondary effect. However, this review revisits a group of neoplasias [subgroups of paragangliomas, gastrointestinal stromal tumours (GISTs) and renal carcinomas] in which dysfunction of the mitochondrial complex 2 is the primary cause of neoplasia. The importance of recognising these tumours is emphasised because of their strong hereditary basis and unique natural histories. Immunohistochemistry for SDHB has emerged as vital tool in the definitive diagnosis of these specific examples of mitochondrial driven neoplasia.
PHAEOCHROMOCTYOMAS AND PARAGANGLIOMAS ARE STRONGLY HEREDITARY Phaeochromocytomas and paragangliomas are rare tumours with a combined estimated annual clinical incidence of 3 per million.4 By convention, tumours arising within the adrenal medulla (the largest paraganglion in the body) are known as phaeochromocytomas, whilst histologically identical tumours arising elsewhere are termed paragangliomas.5 Rather than enforcing this artificial separation, phaeochromocytomas and paragangliomas can be considered one united group of tumours, hereby known as PHEO/PGL. In contrast to the classic maxim of 10%, it is now estimated that at least 30% of PHEO/PGL are hereditary.6 To date, autosomal dominant germline mutations in 12 tumour suppressor genes have been associated with PHEO/PGL and there
2012 Royal College of Pathologists of Australasia
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remain familial cases for which the causative genes are currently unknown. At the time of writing, these genes include RET, VHL, NF1, TMEM127, MAX, KIF1Bb, PHD2, SDHA, SDHB, SDHC, SDHD and SDHAF2.6–11 Familial PHEO/PGL is often not recognised clinically due to incomplete penetrance, phenotypic heterogeneity, lack of clinical awareness and, in the case of SDHD mutation, parent of origin effect masking family histories. In fact, in PHEO/PGL which appear to be sporadic based on the absence of a family history, the rate of occult germline mutation is said to be about 12% and ranges from 7.5% to 24%.12–14 Neurofibromatosis should be clinically apparent as the classic cutaneous features are always present before PHEO/PGL presents, however the other syndromes are commonly clinically silent. Therefore, it has been recommended that genetic testing be considered if not performed for some, most or all of the aforementioned genes whenever a PHEO/PGL is encountered, even if it appears to be sporadic.6 Clinicians and pathologists are well aware of multiple endocrine neoplasia type 2 (MEN2), von Hippel–Lindau syndrome (VHL) and neurofibromatosis type 1 (NF1) because of the constellation of classic tumours with which they are associated. Unfortunately, mutations of SDHA, SDHB, SDHC, SDHD and SDHAF2 are currently under-recognised which is particularly unfortunate given that, as a group, they account for up to 15% of all cases of PHEO/PGL6 (that is half of all familial cases). The SDHA, SDHB, SDHC and SDHD genes are autosomal but encode for proteins which are assembled in the mitochondria to form the mitochondrial complex 2, also known as succinate dehydrogenase/succinate-ubiquinone oxireductase.6 The mitochondrial complex 2 is a key respiratory enzyme linking the Krebs cycle and electron transport chain. Illustrated schematically in Fig. 1, SDHC and SDHD comprise the anchoring component which attaches the complex to the inner mitochondrial membrane, whereas SDHA and SDHB comprise the catalytic component of the mitochondrial complex 2. SDHAF2 is required for insertion of the FAD co-factor into SDHA. When any component of the mitochondrial complex 2 undergoes double hit inactivation, it appears that the entire complex becomes unstable, resulting in degradation of the SDHB
SDHC
Electron transport chain
SDHD
Inner mitochondrial membrane
Co Q
III
Cyt C IV
SDHB
V
SDHA ADP
e− Succinate
ATP
Fumarate KREBS cycle
Fig. 1 The mitochondrial complex 2 is composed of the anchoring component (SDHC and SDHD) and the catalytic component (SDHA and SDHB). SDHAF2 is required for insertion of the FAD co-factor into SDHA. Whenever any component undergoes double hit inactivation, immunohistochemistry for SDHB becomes negative.
subunit. Therefore, immunohistochemistry for SDHB becomes negative whenever there is double hit mutation/inactivation of SDHA, SDHB, SDHC, SDHD or SDHAF2. In contrast to most other double hit models of tumourigenesis, double hit inactivation almost always occurs in the presence of a germline mutation rather than being due to two somatic events. In fact we are only aware of two reports of double hit SDHx inactivation in the absence of germline mutation.15–17 As a result, negative immunohistochemical staining for SDHB occurs whenever there is germline mutation of SDHA, SDHB, SDHC or SDHD, and negative staining for SDHB is now validated as a highly sensitive marker for germline mutation of any of the SDH subunits.6,12 When SDHB immunohistochemistry was first described, approximately 10% of individuals with PHEO/PGL who were thought to be wild type for all mutations were found to be SDHB negative and many of these clearly had hereditary or syndromic features, suggesting that immunohistochemistry for SDHB may be more sensitive for hereditary or syndromic disease that conventional genetic analyses.6,12 It was only after these first reports that germline mutation of SDHAF2 (previously called SDH5) and SDHA were shown to be associated with PHEO/PGL.18,19 The PHEO/PGL associated with these mutations have now been shown to also demonstrate negative staining for SDHB.20 That is, these hereditary tumours were identified by negative staining for SDHB even before the causative genes were identified. Given that mammalian mitochondria contain 1100 proteins, of which nearly 300 are uncharacterised,19 this raises the possibility that there may still be other as yet undescribed genes associated with mitochondrial dysfunction, tumourigenesis and negative staining for SDHB. For example, the Carney triad is the syndromic but not hereditary association of gastric epithelioid GIST, paraganglioma and pulmonary chondroma. Although it is clearly syndromic, the molecular cause is currently unknown. However the PHEO/PGL associated with the Carney triad have also been demonstrated to show negative staining for SDHB indicating that these tumours are in some way related to mitochondrial complex 2 dysfunction.21 In conclusion, it appears that negative staining for SDHB is currently more sensitive than molecular methods for detecting syndromic PHEO/PGL driven by mitochondrial dysfunction. It is recommended that all PHEO/PGL undergo immunohistochemistry for SDHB, which can be performed on archived, formalin fixed, paraffin embedded tumour blocks using a commercially available mouse monoclonal antibody. Because it represents a phenotype rather than a genotype test, we do not believe that formal genetic counselling is necessary before immunohistochemistry is performed (an approach which is highly analogous to screening for Lynch syndrome by first performing immunohistochemistry for DNA mismatch repair proteins). However we do emphasise the need for genetic counselling and specific informed consent before definitive formal genetic testing is begun. The yield of SDHDB as a screening test is particularly high in paraganglioma (extra-adrenal PHEO/PGL) where approximately 40% of unselected consecutive tumours will show negative staining.6 Immunohistochemistry for SDHB has a much lower yield in adrenal phaeochromocytoma where perhaps as few as 3% of tumours show negative staining,6 reflecting the fact that mitochondrial complex 2 mutation is a much rarer cause of adrenal phaeochromocytoma than extraadrenal paraganglioma.
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SDH AND MITOCHONDRIAL DRIVEN NEOPLASIA
If a tumour shows negative staining for SDHB, it should also undergo immunohistochemistry for SDHA. Tumours which are associated with germline mutation of SDHA have now been shown to invariably demonstrate negative staining for SDHA and SDHB, whereas tumours associated with germline mutation of SDHB, SDHC or SDHD will show positive staining for SDHA.20 Therefore, negative staining for SDHA can be considered prima facie evidence of germline SDHA mutation and confirmatory genetic testing is then offered. For SDHB negative but SDHA positive tumours, germline SDHA mutation can be considered excluded and sequential testing for SDHB, SDHC and SDHD is offered with the order of testing being based on the relative frequencies of pathogenic mutations and associated clinical findings at each anatomical site (summarised in Table 1). Generally for intra-abdominal, extra-adrenal tumours, SDHB is tested first, thence SDHD and finally SDHC (which is considerably less common than SDHB and SDHD mutation). For head and neck tumours SDHD is tested first, then SDHB and finally SDHC. If no mutations are found, the possibility of syndromic but non-hereditary disease (the Carney triad) or mutations in rarer (SDHAF2) or unknown genes should be considered. Several international groups have demonstrated that immunohistochemistry for SDHB is a robust and reliable method to screen for SDH mutations. However, we caution about the need for care and experience in interpreting SDHB immunohistochemistry. Positive staining is characterised by a granular cytoplasmic pattern of staining (a mitochondrial pattern). Negative staining refers to the complete absence of this mitochondrial pattern of staining in all neoplastic cells. This approach to requiring a positive internal control before a case is considered negative (a significant result) is highly analogous to the more widespread approach of screening for Lynch syndrome using immunohistochemistry for DNA mismatch repair associated proteins.22 Only cases which are completely negative, yet maintain a positive internal control in non-neoplastic cells, are considered informative (Fig. 2). Cases with a diffuse cytoplasmic blush which lack the distinctive granularity of mitochondrial staining are considered negative. This negative pattern of staining with a non-specific cytoplasmic blush occurs more commonly with SDHD mutation than SDHB or SDHC mutation.6 It may be difficult to distinguish this non-specific cytoplasmic blush from true positive staining and SDHB immunohistochemistry is best interpreted by an experienced observer with access to the quality assurance provided by follow-up genetic testing. Table 1
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Fig. 2 Negative staining for SDHB in the neoplastic cells of a paraganglioma associated with germline mutation of one of the components of the mitochondrial complex 2. Note the positive (granular cytoplasmic) staining in the nonneoplastic endothelial cells, lymphocytes, smooth muscle cells and sustentacular cells. The presence of such an internal positive control is essential before a stain is considered negative.
SDHB MUTATED PHEO/PGL DEMONSTRATE A HIGH RATE OF MALIGNANT BEHAVIOUR Currently malignant PHEO/PGL is only diagnosed in the presence of metastases.23 Because of the propensity for a syndromic presentation and therefore for multifocal disease, the definition of malignant behaviour in PHEO/PGL is problematic and currently a metastasis is defined by the presence of tumour cells at a site where paraganglion tissue is not normally found.23 For example, a para-aortic deposit of tumour which may conceptually represent a metastasis from a previously resected phaeochromocytoma or a second primary tumour, is not considered evidence of malignant behaviour because the para-aortic region is a common site for paraganglioma. Historically, different investigators have employed different criteria for malignancy and some investigators have employed different criteria for malignancy for adrenal phaeochromocytomas versus extra-adrenal paraganglioma, making the true incidence of malignant behaviour difficult to estimate. With this caveat, it appears that the classic maxim of tens still applies to the risk of malignant behaviour for PHEO/PGL as a group with the quoted rate of metastasis usually being given as around 10%.23 Importantly, the rate of malignant behaviour of PHEO/ PGL found to be associated with SDHB mutation is very high. For example for SDHB mutation carriers, the estimated risk of developing a malignant PHEO/PGL (defined by the presence of
Clinical features of SDHB, SDHC and SDHD mutated PHEO/PGL
Syndrome
Gene
Clinical syndrome
Paraganglioma syndrome type 1 (PGL1)
SDHD(11q23)
Paraganglioma syndrome type 3 (PGL3) Paraganglioma syndrome type 4 (PGL4)
SDHC(1q21-23) SDHB(1p35-36)
Phaeochromocytomas/paragangliomas Most common locations: 1. Head and neck 2. Adrenal 3. Intra-abdominal extra-adrenal 4. Thorax Head and neck paragangliomas (especially carotid body tumours) Phaeochromocytomas/paragangliomas Increased risk of malignant behaviour Most common locations: 1. Intraabdominal extra-adrenal 2. Adrenal 3. Head and neck 4. Thorax
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metastases) by the age of 70 is at least 30%.24 Furthermore, approximately 30% of malignant PHEO/PGL will be shown to be associated with SDHB mutation and this rises to 72% if paediatric tumours are studied.25 Therefore, it is particularly important to recognise paraganglioma associated with germline SDHB mutation because of the very high rate of malignant behaviour, and all SDHB mutated PHEO/PGL should be considered at high risk for metastasis and recurrence regardless of other morphological or clinical features.
SUCCINATE DEHYDROGENASE (SDH) DEFICIENT GIST For some time it has been recognised that the great majority of gastrointestinal stromal tumours (GISTs) occurring in childhood are quite different to the great majority of GISTs occurring in adults.26–28 These tumours, until recently known as ‘paediatric wild type GISTs’, are wild type for KIT and PDGFRA and demonstrate quite distinctive morphology and clinical features. For example, they always arise in the stomach, demonstrate a multinodular growth pattern and are commonly multicentric, have a prognosis not predicted by size and mitotic rate and very frequently metastasise to lymph nodes. They do not respond to imatinib, but may respond better to sunitinib. The GISTs arising in the Carney triad show identical morphology and clinical features to these paediatric wild type GISTs.29 Using a comparative genomic hybridisation (CGH) approach, the paediatric wild type GISTs and the GISTs of the Carney triad were shown to hybridise together and quite separately to usual adult GIST or the rare cases of paediatric GIST associated with KIT or PDGFRA mutations.26 That is, paediatric wild type GISTs and the GISTs which arise in the Carney triad are a unique clinicopathological entity. Given that the Carney triad demonstrates incomplete penetrance and some individuals may present first with GIST in adulthood decades before other manifestations of the syndrome arise (if they arise at all), there must also be many cases of these unique GISTs occurring in adults which have been unrecognised until now. In 2010 we reported that negative staining for SDHB is characteristic of the GISTs of the Carney triad and the subgroup of paediatric GISTs they resemble (that is, paediatric wild type GISTs) and also that when negative staining occurs in apparently sporadic GISTs in adults, these GISTs show Table 2
Comparison of SDHB positive (usual adult) and SDHB deficient GISTs
Location KIT/PDGFRA mutation Germline SDH mutation Sex incidence Usual age Prognosis predicted by size and mitotic rate Multifocality Predominant cell Lymph node metastasis Behaviour of metastases Imatinib response Syndromic presentation Associated syndromes
* {
morphological and clinical features similar to paediatric and Carney triad GISTs but different to usual adult GISTs.21 The combined results of several studies have now confirmed that negative staining for SDHB identifies a unique subtype of GIST for which the terminology ‘SDH deficient GIST’ has now been proposed.21,30,31–34 The concept of the SDH deficient GIST unites the GISTs of the Carney triad, paediatric wild type GISTs, the GISTs associated with Carney–Stratakis syndrome (the hereditary association of GIST and paraganglioma due to germline SDH mutation) and a fraction of apparently sporadic adult wild type GISTs into a single distinct type of tumour with identical morphology and clinical features. The unique clinical, morphological and genetic features of SDH deficient GISTs are summarised in Table 2 and illustrated in Fig. 3 and 4.21,30,31,33,34 Briefly, they always arise in the stomach, commonly demonstrate a multilobulated or multinodular growth pattern (often with a nested or almost paraganglioma-like arrangement of cells), frequently metastasise to lymph nodes, are always wild type for KIT and PDGFRA and do not respond to imatinib but may respond to sunitinib. The prognosis of SDH deficient GISTs cannot be predicted by size and mitotic rate (that is, even small, mitotically inactive SDH deficient GISTs may metastasise). However, when metastases do occur they may be strikingly indolent, sometimes remaining stable for years or decades. SDH deficient GISTs are particularly common in childhood and young adulthood. Importantly SDH deficient GISTs also account for between 5% and 7.5% of all gastric GISTs occurring in adults.21,32 When they occur in adulthood their unique morphological and clinical features are usually not recognised. Therefore, it is recommended that immunohistochemistry for SDHB should be performed on gastric GISTs with compatible morphology. Clues to the diagnosis include a predominantly epithelioid morphology, multifocality or multinodularity and lymph node metastases. SDH deficient GISTs appear to always be positive for KIT and DOG1 by immunohistochemistry, in contrast to PDGFRA mutated gastric GISTs which may also demonstrate an epithelioid morphology but are commonly negative for KIT. We would normally perform SDHB immunohistochemistry before sequencing for KIT and PDGFRA, however an alternative approach would be to perform SDHB immunohistochemistry on all wild type GISTs because SDHB negative GISTs are invariably wild type for
SDHB positive
SDHB negative
Entire GIT (very occasionally extra-GIT) Usually (>90%) No Equal Older adult Yes Rare Spindled Rare or never Aggressive Usual Very rare Neurofibromatosis1 Germline KIT mutation Germline PDGFRA
Stomach only Never Sometimes Females more common than male Commonly child or young adult No Common Epithelioid Common Indolent Absent More common but still minority Carney triad* Carney–Stratakis/PGL syndromes{
The Carney triad is the non-hereditary association of GIST, paraganglioma and pulmonary chondroma. The molecular mechanism is unknown. Carney–Stratakis syndrome/PGL syndromes are the hereditary association of paraganglioma and GIST due to germline SDH mutations.
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SDH AND MITOCHONDRIAL DRIVEN NEOPLASIA
Fig. 3 Serially (A) H&E and (B) SDHB stained whole mount sections of a metastasis to the liver of an SDH deficient GIST. The intense staining of the mitochondria rich hepatocytes contrasts strongly with the completely negative staining of the GIST.
these two genes. Although the great majority of paediatric wild type GISTs will be SDHB negative,33 in adults it appears that only approximately one-quarter to one-third of all wild type GISTs (perhaps half of gastric wild type GISTs) will be of the SDH deficient type.21,30,32 The genetic mechanisms of neoplasia in SDH deficient GISTs is not clear in all cases. Working with a predominantly paediatric population, Janeway et al. tested for germline mutation of SDHB, SDHC and SDHD in 34 SDH deficient GISTs (without a personal or family history of paraganglioma) and found germline mutations in SDHB and SDHC in four cases (12%).33 In contrast, working with a predominantly adult population, Miettinen et al. found no germline mutations of SDHB, SDHC or SDHD in 10 cases studied.32 Because SDHA has only recently been recognised as a true tumour suppressor gene, neither of these two groups looked for SDHA mutation. However, working in a different cohort at a similar time, Pantaleo et al. have now documented SDHA mutation in four GISTs.35,36 As a significant proportion of SDH deficient GISTs are associated with SDHA, SDHB, SDHC or SDHD mutation, it is recommended that individuals with SDH deficient GISTs be offered formal genetic testing. If the immunohistochemical profile of GISTs is similar to PHEO/PGL it would be assumed that these SDHA mutated GISTs would be SDHA and
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SDHB negative by immunohistochemistry, whereas other SDH deficient GISTs would be SDHA positive but SDHB negative. Therefore, immunohistochemistry for both SDHB and SDHA may help to triage the order in which these genes are tested. However, this hypothesis has not been tested to date. GISTs arising in the setting of neurofibromatosis, although often wild type and sometimes occurring in childhood, have been consistently shown to demonstrate positive staining for SDHB, occur throughout the gastrointestinal tract and display a predominantly spindled morphology.21,32,33,37 Although they may sometimes present as paediatric wild type GISTs (or at least GISTs which arise in childhood and are wild type for KIT and PDGFRA), they are not SDH deficient GISTs. In summary the combined results of several studies indicate that germline mutation of SDHA, SDHB, SDHC or SDHD may be the cause of at least some SDH deficient GISTs and that genetic testing for each of these genes should be offered whenever an SDH deficient GIST is recognised. However, it appears that in contrast to SDHB negative paraganglioma (where the majority of SDHB negative cases will be shown to be associated with a germline mutation), the majority of SDHB negative GISTs do not harbour a germline mutation in any of the recognised components of the mitochondrial complex 2. Some of these individuals will have the Carney triad (the molecular pathogenesis of which is unknown but clearly involves dysfunction of the mitochondrial complex 2), but the majority will not. The mechanism of mitochondrial complex 2 dysfunction and tumourigenesis in these cases which are not associated with germline SDH mutation therefore is unknown. In the interim, we recommend that in addition to genetic testing for SDHA, SDHB, SDHC and SDHD, individuals diagnosed with SDH deficient GIST should be offered clinical screening and follow-up for paraganglioma and pulmonary chondroma as well as for recurrent or metachronous GIST. In addition, consideration should be given to the unique natural history of these tumours when planning treatment and follow-up.
SDHB NEGATIVE RENAL CARCINOMA Approximately 4% of renal carcinomas are hereditary and there are known associations with germline mutations of VHL, c-MET, FH and FLCN and constitutional translocations of chromosome 3.38 Germline SDHB, SDHC and SDHD mutations have now also been clearly associated with renal neoplasia.24,39 Historically these tumours have usually been classified as carcinoma (commonly conventional clear cell renal carcinoma but also chromophobe carcinoma, papillary renal carcinoma or other types) but sometimes these tumours have been classified as oncocytoma (summarised in40). In 2011
Fig. 4 Serially (A) H&E and (B) SDHB stained whole sections of a metastasis to the liver of an SDH deficient GIST. At this magnification the distinctive epithelioid morphology and somewhat nested architecture of the GIST is noted. The positive staining for SDHB in the non-neoplastic hepatocytes, endothelial and perivascular cells is again noted.
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Fig. 5 (A–D) Three of the four SDHB associated renal carcinomas we reported demonstrated similar histological features. The most striking feature and easily recognisable feature is the presence of intracytoplasmic inclusions which contain either (C) eosinophilic or (D) vacuolated material.
we demonstrated that negative staining for SDHB identifies renal neoplasia associated with germline SDHB mutation.40,41 This finding has since been confirmed by others.42 If what is known about the staining profile of PHEO/PGL can be extrapolated to renal carcinoma, it is likely that tumours associated with SDHC and SDHD mutation will also be shown to be negative for SDHB by immunohistochemistry. Indeed, recently immunohistochemistry for SDHB has been reported as being negative in one case of renal carcinoma arising in the setting of SDHC mutation.39 However, given that SDHB staining was positive in another renal carcinoma with a different morphology arising in the same patient, the hypothesis that negative staining for SDHB will also identify SDHC and SDHD mutated renal carcinomas clearly needs to be tested further before it is put into practice. It is interesting to note that in most, if not all, of the early reports of renal neoplasia arising in the setting of SDH mutation, the morphology of the tumours was not illustrated or described. When we examined a series of four renal tumours arising in the setting of germline SDHB mutation, we found that three of them were characterised by unique and distinctive morphological features (Fig. 5).40 Briefly, they were composed of cuboidal cells with bubbly eosinophilic cytoplasm and indistinct cell borders, often arranged in a nested architecture.
Many of the cells displayed distinctive cytoplasmic inclusions which were vacuolated or contained pale eosinophilic material. However, one of the tumours we encountered did not show this stereotypical morphology and demonstrated sarcomatoid and papillary areas (Fig. 6). It would have been unrecognisable if not for immunohistochemistry for SDHB. In summary, the morphological clues to SDHB associated renal neoplasia are real but subtle and clearly not present in all tumours. Therefore, immunohistochemistry for SDHB should be used to screen for SDH mutations, not only whenever these morphological clues are encountered, but also when there are clinical red flags for hereditary renal neoplasia. These clinical red flags include bilaterality, multifocality, young age of onset, or a personal or family history of either renal carcinoma or PHEO/PGL (particularly if von Hippel–Lindau syndrome has been excluded) or GIST.
OTHER TUMOURS POTENTIALLY ASSOCIATED WITH SDH DYSFUNCTION Although the link between SDHB mutation and neuroblastoma has been disputed and if such an association does occur it is clearly rare,43,44 at least three individuals with germline SDH mutation and neuroblastoma, including one who subsequently
Fig. 6 (A,B) In this example of SDHB associated renal carcinoma, the neoplastic cells are undifferentiated and there are sarcomatoid and papillary areas. (B) At higher power some very subtle cytoplasmic vacuolation (perhaps corresponding to the inclusions illustrated in Fig. 5) are present. Immunohistochemistry for SDHB was negative, illustrating the importance of performing this stain if there are clinical red flags, even if the stereotypical morphology is absent.
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SDH AND MITOCHONDRIAL DRIVEN NEOPLASIA
developed renal carcinoma, have been reported.45 Despite the rarity of the association, there may yet be a role for screening neuroblastomas for germline mutations with SDHB immunohistochemistry. A single case of an adrenal adenomatoid tumour in association with a germline SDHD mutation has been reported.46 There has also been a single growth hormone producing pituitary adenoma reported in association with SDHD mutation.47 Both these tumours were reported to show negative staining for SDHB by immunohistochemistry, supporting the argument that they are bona fide manifestations of SDHD germline mutations rather than incidental findings. However, further investigation is required before these novel associations can be considered definitive. Again immunohistochemistry for SDHB appears to be an ideal screening tool for assessing the relationship of these tumours with mitochondrial complex 2 dysfunction.
MECHANISM OF NEOPLASIA IN MITOCHONDRIAL COMPLEX 2 DYSFUNCTION The mechanisms by which mitochondrial complex 2 dysfunction causes neoplasia is not fully understood but the key events are gradually becoming apparent (reviewed in 48). Hypoxia inducible factors (HIFs) are sequence specific DNA-binding transcription factors that facilitate cell adaptation and survival in hypoxic conditions. Both mitochondrial complex deficiency and VHL mutation lead to stabilisation of HIFs. In turn the HIFs induce transcription of several genes involved in angiogenesis, energy metabolism, survival and growth. The downstream cellular events which occur in both mitochondrial complex 2 deficiency and VHL mutation therefore are very similar to those associated with hypoxia and these events have been termed a ‘pseudohypoxic response’. In contrast, the transcription profile displayed by RET, NF1, KIF1BB, TMEM127 and MAX related tumours are quite different and linked to the activation of tyrosine kinase pathways. Although the exact mechanism by which pseudohypoxia results in neoplasia is unclear, the link between PHEO/PGL tumourigenesis and cellular events mimicking hypoxia is reinforced by the remarkable but longstanding observation that individuals exposed to chronic hypoxia by living at high altitude are predisposed to paraganglioma compared to those living at sea level (summarised in 49).
CONCLUSION PHEO/PGL is a highly hereditary condition. Although only approximately 10% of patients have a positive family history, currently 30% will be associated with germline mutation and this figure may rise as more genes are described. Therefore, any PHEO/PGL should be considered potentially hereditary until this possibility is excluded. By identifying the approximately 15% of PHEO/PGL associated with mitochondrial complex 2 dysfunction, immunohistochemistry for SDHB is a vital tool for triaging genetic testing of these tumours and the yield is particularly high in extra-adrenal PHEO/PGL. The high rate of malignant behaviour of SDHB mutated PHEO/PGL is emphasised and recognition of SDHB mutation should lead to more aggressive surgery and surveillance, particularly compared to tumours arising in MEN2 and von Hippel–Lindau syndrome which are more commonly bilateral but have a low risk of metastasis. In contrast to SDHB negative renal tumours and PHEO/PGL, it appears that only a small proportion of SDH deficient GISTs
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will be associated with germline SDH mutation; others will be associated with the Carney triad but at this stage it appears that most are sporadic. The mechanism of neoplasia in these SDH deficient GISTs not associated with SDH mutation is unknown. Although we have been unable to recognise morphological differences between SDH mutated and non-mutated PHEO/ PGL, SDH deficient GISTs and a subgroup of SDHB mutated renal carcinomas show very characteristic morphological and clinical features which, if recognised, can lead to immunohistochemistry for SDHB and therefore definitive diagnosis. Early diagnosis of SDH related neoplasia is clearly advantageous in view of the distinct natural history of these tumours and the potential for genetic testing and screening to alleviate the burden of disease in these kindreds. In conclusion, after all these years it appears that Warburg was right. Some tumours (albeit relatively rare subsets of PHEO/PGL, GIST and renal carcinoma and perhaps a few other tumour types) are indeed driven primarily by mitochondrial complex 2 dysfunction. Surgical pathologists should be aware of this association and the fact that immunohistochemistry for SDHB allows definitive identification of these tumours. Conflicts of interest and sources of funding: The author declares that there is no conflict of interest. Address for correspondence: Dr A. J. Gill, Department of Anatomical Pathology, Royal North Shore Hospital, Pacific Highway, St Leonards, NSW 2065, Australia. E-mail: [email protected]
References 1. Koppenol WH, Bounds PL, Dang CV. Otto Warburg’s contributions to current concepts of cancer metabolism. Nat Rev Cancer 2011; 11: 325–37. 2. Tennant DA, Dura´n RV, Gottlieb E. Targeting metabolic transformation for cancer therapy. Nat Rev Cancer 2010; 10: 267–77. 3. Warburg O. On the origin of cancer cells. Science 1956; 123: 309–14. 4. Baysal BE. Hereditary paraganglioma targets diverse paraganglia. J Med Genet 2002; 39: 617–22. 5. DeLellis RA, Lloyd RV, Heitz RV, Heng C, editors. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Endocrine Organs. Lyon: IARC Press, 2004. 6. Gill AJ, Benn DE, Chou A, et al. Immunohistochemistry for SDHB triages genetic testing of SDHB, SDHC, and SDHD in paragangliomaphaeochromocytoma syndromes. Hum Pathol 2010; 41: 805–14. 7. Comino-Mendez I, Gracia-Aznarez FJ, Schiavi F, et al. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nat Genet 2011; 43: 663–7. 8. Yeh IT, Lenci RE, Qin Y, et al. A germline mutation of the KIF1B gene on 1p36 in a family with neural and nonneural tumors. Hum Genet 2008; 124: 279–85. 9. Ladroue C, Carcenac R, Leporrier M, et al. PHD2 mutation and congenital erythrocytosis with paraganglioma. N Engl J Med 2008; 359: 2685–92. 10. Burnichon N, Brie`re JJ, Libe´ R, et al. SDHA is a tumor suppressor gene causing paraganglioma. Hum Mol Genet 2010; 19: 3011–20. 11. Hensen EF, Bayley JP. Recent advances in the genetics of SDH-related paraganglioma and pheochromocytoma. Fam Cancer 2011; 10: 355–63. 12. van Nederveen FH, Gaal J, Favier J, et al. An immunohistochemical procedure to detect patients with paraganglioma and phaeochromocytoma with germline SDHB, SDHC or SDHD gene mutations: a retrospective and prospective analysis. Lancet Oncol 2009; 8: 764–71. 13. KorperShoek E, van Nederveen FH, Danenberg H, et al. Genetic analysis of apparently sporadic pheochromocytomas: The Rotterdam Experience. Ann NY Acad Sci 2006; 1073: 138–48. 14. Neuman HP, Bausc B, McWhinney SR, et al. Germ-line mutations in nonsyndromic pheochromocytoma. N Eng J Med 2002; 346: 1459–66. 15. van Nederveen FH, Korpershoek E, Lenders JW, et al. Somatic SDHB mutation in extraadrenal pheochromocytoma. N Engl J Med 2007; 357: 306–8. 16. Bayley JP, van Minderhout I, Weiss MM, et al. Mutation analysis of SDHB and SDHC novel germline mutations in sporadic head and neck
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REVIEW
Succinate dehydrogenase (SDH) and mitochondrial driven neoplasia ANTHONY J. GILL*{ *Department of Anatomical Pathology and Northern Cancer Translational Research Unit, Royal North Shore Hospital, St Leonards, and {University of Sydney, Sydney, New South Wales, Australia
Summary The genes for the succinate dehydrogenase (SDH) subunits SDHA, SDHB, SDHC and SDHD are encoded in the autosome. The proteins are assembled in the mitochondria to form the mitochondrial complex 2, a key respiratory enzyme which links the Krebs cycle and the electron transport chain. Thirty percent of phaeochromocytoma and paraganglioma (PHEO/PGL) are hereditary and perhaps as many as half of these familial cases are caused by germline mutations of the SDH subunits. Negative immunohistochemical staining for the SDHB subunit identifies PHEO/PGL associated with germline mutation of any of the mitochondrial complex 2 components and can be used to triage formal genetic testing of all PHEO/PGL for SDH mutations. PHEO/PGL associated with SDHA mutation also show negative staining for SDHA as well as SDHB. A unique subgroup of gastrointestinal stromal tumours (GISTs) are driven by mitochondrial complex 2 dysfunction. These SDH deficient GISTs can also be definitively identified by negative staining for SDHB and show distinct clinical and morphological features including frequent onset in childhood and young adulthood, gastric location, a tendency to multifocality, absence of KIT and PDGFRA mutations, a prognosis not predicted by size and mitotic rate and a tendency to indolent behaviour of metastases. Some of these SDH deficient GISTs are driven by classical SDH mutations, but the precise mechanisms of tumourigenesis in many (including those associated with the Carney triad) remain unknown. Germline SDHB mutation is associated with a newly recognised type of renal carcinoma which commonly but not always demonstrates distinctive morphology and can also be recognised by negative staining for SDHB. Immunohistochemistry for SDHB therefore has emerged as a useful tool to recognise these distinct neoplasias driven by mitochondrial complex 2 dysfunction and to triage formal genetic testing for the associated syndromes. Abbreviations: GIST, gastrointestinal stromal tumour; PHEO/PGL, phaeochromocytoma and paraganglioma; SDH, succinate dehydrogenase. Key words: Gastrointestinal stromal tumour, GIST, paraganglioma, phaeochromocytoma, SDHB. Received 23 February, revised 7 March, accepted 12 March 2012
INTRODUCTION Otto Heinrich Warburg (1883–1970) was awarded the 1931 Nobel prize in medicine or physiology for his groundbreaking work on the nature and mechanisms of action of the respiratory enzymes.1 He is better known now for his demonstration that even in the presence of abundant oxygen most cancer cells Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0b013e3283539932
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produce energy predominantly by glycolysis (resulting in lactate production) rather than by the far more efficient process of mitochondrial oxidative respiration on which most nonneoplastic cells rely. The Warburg effect, as this phenomenon and the subsequent demand for large amounts of glucose by malignant cells has become known, has been a poorly understood but consistently observed occurrence in malignant cells. It is the basis of the routine detection and monitoring of human malignancies by fluorodeoxyglucose positron emission tomography (PET scan) and the development of agents which target the ‘addiction’ of malignant cells to glucose is an area of much promising research in the field of drug development.2 However, Warburg went further than to merely observe the link between deficient mitochondrial oxidative respiration and neoplasia. He steadfastly maintained that defective respiration caused malignancies. As he wrote in 1956: ‘there is only one common cause into which all other causes of cancer merge, the irreversible injuring of respiration.’3 Warburg’s belief that altered mitochondrial respiration is the primary cause of cancer (the Warburg hypothesis) has been surpassed by the understanding that malignancies are caused primarily by DNA mutations which lead to growth dysregulation and genomic instability. Mitochondrial dysfunction appears to be a secondary effect. However, this review revisits a group of neoplasias [subgroups of paragangliomas, gastrointestinal stromal tumours (GISTs) and renal carcinomas] in which dysfunction of the mitochondrial complex 2 is the primary cause of neoplasia. The importance of recognising these tumours is emphasised because of their strong hereditary basis and unique natural histories. Immunohistochemistry for SDHB has emerged as vital tool in the definitive diagnosis of these specific examples of mitochondrial driven neoplasia.
PHAEOCHROMOCTYOMAS AND PARAGANGLIOMAS ARE STRONGLY HEREDITARY Phaeochromocytomas and paragangliomas are rare tumours with a combined estimated annual clinical incidence of 3 per million.4 By convention, tumours arising within the adrenal medulla (the largest paraganglion in the body) are known as phaeochromocytomas, whilst histologically identical tumours arising elsewhere are termed paragangliomas.5 Rather than enforcing this artificial separation, phaeochromocytomas and paragangliomas can be considered one united group of tumours, hereby known as PHEO/PGL. In contrast to the classic maxim of 10%, it is now estimated that at least 30% of PHEO/PGL are hereditary.6 To date, autosomal dominant germline mutations in 12 tumour suppressor genes have been associated with PHEO/PGL and there
2012 Royal College of Pathologists of Australasia
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remain familial cases for which the causative genes are currently unknown. At the time of writing, these genes include RET, VHL, NF1, TMEM127, MAX, KIF1Bb, PHD2, SDHA, SDHB, SDHC, SDHD and SDHAF2.6–11 Familial PHEO/PGL is often not recognised clinically due to incomplete penetrance, phenotypic heterogeneity, lack of clinical awareness and, in the case of SDHD mutation, parent of origin effect masking family histories. In fact, in PHEO/PGL which appear to be sporadic based on the absence of a family history, the rate of occult germline mutation is said to be about 12% and ranges from 7.5% to 24%.12–14 Neurofibromatosis should be clinically apparent as the classic cutaneous features are always present before PHEO/PGL presents, however the other syndromes are commonly clinically silent. Therefore, it has been recommended that genetic testing be considered if not performed for some, most or all of the aforementioned genes whenever a PHEO/PGL is encountered, even if it appears to be sporadic.6 Clinicians and pathologists are well aware of multiple endocrine neoplasia type 2 (MEN2), von Hippel–Lindau syndrome (VHL) and neurofibromatosis type 1 (NF1) because of the constellation of classic tumours with which they are associated. Unfortunately, mutations of SDHA, SDHB, SDHC, SDHD and SDHAF2 are currently under-recognised which is particularly unfortunate given that, as a group, they account for up to 15% of all cases of PHEO/PGL6 (that is half of all familial cases). The SDHA, SDHB, SDHC and SDHD genes are autosomal but encode for proteins which are assembled in the mitochondria to form the mitochondrial complex 2, also known as succinate dehydrogenase/succinate-ubiquinone oxireductase.6 The mitochondrial complex 2 is a key respiratory enzyme linking the Krebs cycle and electron transport chain. Illustrated schematically in Fig. 1, SDHC and SDHD comprise the anchoring component which attaches the complex to the inner mitochondrial membrane, whereas SDHA and SDHB comprise the catalytic component of the mitochondrial complex 2. SDHAF2 is required for insertion of the FAD co-factor into SDHA. When any component of the mitochondrial complex 2 undergoes double hit inactivation, it appears that the entire complex becomes unstable, resulting in degradation of the SDHB
SDHC
Electron transport chain
SDHD
Inner mitochondrial membrane
Co Q
III
Cyt C IV
SDHB
V
SDHA ADP
e− Succinate
ATP
Fumarate KREBS cycle
Fig. 1 The mitochondrial complex 2 is composed of the anchoring component (SDHC and SDHD) and the catalytic component (SDHA and SDHB). SDHAF2 is required for insertion of the FAD co-factor into SDHA. Whenever any component undergoes double hit inactivation, immunohistochemistry for SDHB becomes negative.
subunit. Therefore, immunohistochemistry for SDHB becomes negative whenever there is double hit mutation/inactivation of SDHA, SDHB, SDHC, SDHD or SDHAF2. In contrast to most other double hit models of tumourigenesis, double hit inactivation almost always occurs in the presence of a germline mutation rather than being due to two somatic events. In fact we are only aware of two reports of double hit SDHx inactivation in the absence of germline mutation.15–17 As a result, negative immunohistochemical staining for SDHB occurs whenever there is germline mutation of SDHA, SDHB, SDHC or SDHD, and negative staining for SDHB is now validated as a highly sensitive marker for germline mutation of any of the SDH subunits.6,12 When SDHB immunohistochemistry was first described, approximately 10% of individuals with PHEO/PGL who were thought to be wild type for all mutations were found to be SDHB negative and many of these clearly had hereditary or syndromic features, suggesting that immunohistochemistry for SDHB may be more sensitive for hereditary or syndromic disease that conventional genetic analyses.6,12 It was only after these first reports that germline mutation of SDHAF2 (previously called SDH5) and SDHA were shown to be associated with PHEO/PGL.18,19 The PHEO/PGL associated with these mutations have now been shown to also demonstrate negative staining for SDHB.20 That is, these hereditary tumours were identified by negative staining for SDHB even before the causative genes were identified. Given that mammalian mitochondria contain 1100 proteins, of which nearly 300 are uncharacterised,19 this raises the possibility that there may still be other as yet undescribed genes associated with mitochondrial dysfunction, tumourigenesis and negative staining for SDHB. For example, the Carney triad is the syndromic but not hereditary association of gastric epithelioid GIST, paraganglioma and pulmonary chondroma. Although it is clearly syndromic, the molecular cause is currently unknown. However the PHEO/PGL associated with the Carney triad have also been demonstrated to show negative staining for SDHB indicating that these tumours are in some way related to mitochondrial complex 2 dysfunction.21 In conclusion, it appears that negative staining for SDHB is currently more sensitive than molecular methods for detecting syndromic PHEO/PGL driven by mitochondrial dysfunction. It is recommended that all PHEO/PGL undergo immunohistochemistry for SDHB, which can be performed on archived, formalin fixed, paraffin embedded tumour blocks using a commercially available mouse monoclonal antibody. Because it represents a phenotype rather than a genotype test, we do not believe that formal genetic counselling is necessary before immunohistochemistry is performed (an approach which is highly analogous to screening for Lynch syndrome by first performing immunohistochemistry for DNA mismatch repair proteins). However we do emphasise the need for genetic counselling and specific informed consent before definitive formal genetic testing is begun. The yield of SDHDB as a screening test is particularly high in paraganglioma (extra-adrenal PHEO/PGL) where approximately 40% of unselected consecutive tumours will show negative staining.6 Immunohistochemistry for SDHB has a much lower yield in adrenal phaeochromocytoma where perhaps as few as 3% of tumours show negative staining,6 reflecting the fact that mitochondrial complex 2 mutation is a much rarer cause of adrenal phaeochromocytoma than extraadrenal paraganglioma.
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SDH AND MITOCHONDRIAL DRIVEN NEOPLASIA
If a tumour shows negative staining for SDHB, it should also undergo immunohistochemistry for SDHA. Tumours which are associated with germline mutation of SDHA have now been shown to invariably demonstrate negative staining for SDHA and SDHB, whereas tumours associated with germline mutation of SDHB, SDHC or SDHD will show positive staining for SDHA.20 Therefore, negative staining for SDHA can be considered prima facie evidence of germline SDHA mutation and confirmatory genetic testing is then offered. For SDHB negative but SDHA positive tumours, germline SDHA mutation can be considered excluded and sequential testing for SDHB, SDHC and SDHD is offered with the order of testing being based on the relative frequencies of pathogenic mutations and associated clinical findings at each anatomical site (summarised in Table 1). Generally for intra-abdominal, extra-adrenal tumours, SDHB is tested first, thence SDHD and finally SDHC (which is considerably less common than SDHB and SDHD mutation). For head and neck tumours SDHD is tested first, then SDHB and finally SDHC. If no mutations are found, the possibility of syndromic but non-hereditary disease (the Carney triad) or mutations in rarer (SDHAF2) or unknown genes should be considered. Several international groups have demonstrated that immunohistochemistry for SDHB is a robust and reliable method to screen for SDH mutations. However, we caution about the need for care and experience in interpreting SDHB immunohistochemistry. Positive staining is characterised by a granular cytoplasmic pattern of staining (a mitochondrial pattern). Negative staining refers to the complete absence of this mitochondrial pattern of staining in all neoplastic cells. This approach to requiring a positive internal control before a case is considered negative (a significant result) is highly analogous to the more widespread approach of screening for Lynch syndrome using immunohistochemistry for DNA mismatch repair associated proteins.22 Only cases which are completely negative, yet maintain a positive internal control in non-neoplastic cells, are considered informative (Fig. 2). Cases with a diffuse cytoplasmic blush which lack the distinctive granularity of mitochondrial staining are considered negative. This negative pattern of staining with a non-specific cytoplasmic blush occurs more commonly with SDHD mutation than SDHB or SDHC mutation.6 It may be difficult to distinguish this non-specific cytoplasmic blush from true positive staining and SDHB immunohistochemistry is best interpreted by an experienced observer with access to the quality assurance provided by follow-up genetic testing. Table 1
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Fig. 2 Negative staining for SDHB in the neoplastic cells of a paraganglioma associated with germline mutation of one of the components of the mitochondrial complex 2. Note the positive (granular cytoplasmic) staining in the nonneoplastic endothelial cells, lymphocytes, smooth muscle cells and sustentacular cells. The presence of such an internal positive control is essential before a stain is considered negative.
SDHB MUTATED PHEO/PGL DEMONSTRATE A HIGH RATE OF MALIGNANT BEHAVIOUR Currently malignant PHEO/PGL is only diagnosed in the presence of metastases.23 Because of the propensity for a syndromic presentation and therefore for multifocal disease, the definition of malignant behaviour in PHEO/PGL is problematic and currently a metastasis is defined by the presence of tumour cells at a site where paraganglion tissue is not normally found.23 For example, a para-aortic deposit of tumour which may conceptually represent a metastasis from a previously resected phaeochromocytoma or a second primary tumour, is not considered evidence of malignant behaviour because the para-aortic region is a common site for paraganglioma. Historically, different investigators have employed different criteria for malignancy and some investigators have employed different criteria for malignancy for adrenal phaeochromocytomas versus extra-adrenal paraganglioma, making the true incidence of malignant behaviour difficult to estimate. With this caveat, it appears that the classic maxim of tens still applies to the risk of malignant behaviour for PHEO/PGL as a group with the quoted rate of metastasis usually being given as around 10%.23 Importantly, the rate of malignant behaviour of PHEO/ PGL found to be associated with SDHB mutation is very high. For example for SDHB mutation carriers, the estimated risk of developing a malignant PHEO/PGL (defined by the presence of
Clinical features of SDHB, SDHC and SDHD mutated PHEO/PGL
Syndrome
Gene
Clinical syndrome
Paraganglioma syndrome type 1 (PGL1)
SDHD(11q23)
Paraganglioma syndrome type 3 (PGL3) Paraganglioma syndrome type 4 (PGL4)
SDHC(1q21-23) SDHB(1p35-36)
Phaeochromocytomas/paragangliomas Most common locations: 1. Head and neck 2. Adrenal 3. Intra-abdominal extra-adrenal 4. Thorax Head and neck paragangliomas (especially carotid body tumours) Phaeochromocytomas/paragangliomas Increased risk of malignant behaviour Most common locations: 1. Intraabdominal extra-adrenal 2. Adrenal 3. Head and neck 4. Thorax
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metastases) by the age of 70 is at least 30%.24 Furthermore, approximately 30% of malignant PHEO/PGL will be shown to be associated with SDHB mutation and this rises to 72% if paediatric tumours are studied.25 Therefore, it is particularly important to recognise paraganglioma associated with germline SDHB mutation because of the very high rate of malignant behaviour, and all SDHB mutated PHEO/PGL should be considered at high risk for metastasis and recurrence regardless of other morphological or clinical features.
SUCCINATE DEHYDROGENASE (SDH) DEFICIENT GIST For some time it has been recognised that the great majority of gastrointestinal stromal tumours (GISTs) occurring in childhood are quite different to the great majority of GISTs occurring in adults.26–28 These tumours, until recently known as ‘paediatric wild type GISTs’, are wild type for KIT and PDGFRA and demonstrate quite distinctive morphology and clinical features. For example, they always arise in the stomach, demonstrate a multinodular growth pattern and are commonly multicentric, have a prognosis not predicted by size and mitotic rate and very frequently metastasise to lymph nodes. They do not respond to imatinib, but may respond better to sunitinib. The GISTs arising in the Carney triad show identical morphology and clinical features to these paediatric wild type GISTs.29 Using a comparative genomic hybridisation (CGH) approach, the paediatric wild type GISTs and the GISTs of the Carney triad were shown to hybridise together and quite separately to usual adult GIST or the rare cases of paediatric GIST associated with KIT or PDGFRA mutations.26 That is, paediatric wild type GISTs and the GISTs which arise in the Carney triad are a unique clinicopathological entity. Given that the Carney triad demonstrates incomplete penetrance and some individuals may present first with GIST in adulthood decades before other manifestations of the syndrome arise (if they arise at all), there must also be many cases of these unique GISTs occurring in adults which have been unrecognised until now. In 2010 we reported that negative staining for SDHB is characteristic of the GISTs of the Carney triad and the subgroup of paediatric GISTs they resemble (that is, paediatric wild type GISTs) and also that when negative staining occurs in apparently sporadic GISTs in adults, these GISTs show Table 2
Comparison of SDHB positive (usual adult) and SDHB deficient GISTs
Location KIT/PDGFRA mutation Germline SDH mutation Sex incidence Usual age Prognosis predicted by size and mitotic rate Multifocality Predominant cell Lymph node metastasis Behaviour of metastases Imatinib response Syndromic presentation Associated syndromes
* {
morphological and clinical features similar to paediatric and Carney triad GISTs but different to usual adult GISTs.21 The combined results of several studies have now confirmed that negative staining for SDHB identifies a unique subtype of GIST for which the terminology ‘SDH deficient GIST’ has now been proposed.21,30,31–34 The concept of the SDH deficient GIST unites the GISTs of the Carney triad, paediatric wild type GISTs, the GISTs associated with Carney–Stratakis syndrome (the hereditary association of GIST and paraganglioma due to germline SDH mutation) and a fraction of apparently sporadic adult wild type GISTs into a single distinct type of tumour with identical morphology and clinical features. The unique clinical, morphological and genetic features of SDH deficient GISTs are summarised in Table 2 and illustrated in Fig. 3 and 4.21,30,31,33,34 Briefly, they always arise in the stomach, commonly demonstrate a multilobulated or multinodular growth pattern (often with a nested or almost paraganglioma-like arrangement of cells), frequently metastasise to lymph nodes, are always wild type for KIT and PDGFRA and do not respond to imatinib but may respond to sunitinib. The prognosis of SDH deficient GISTs cannot be predicted by size and mitotic rate (that is, even small, mitotically inactive SDH deficient GISTs may metastasise). However, when metastases do occur they may be strikingly indolent, sometimes remaining stable for years or decades. SDH deficient GISTs are particularly common in childhood and young adulthood. Importantly SDH deficient GISTs also account for between 5% and 7.5% of all gastric GISTs occurring in adults.21,32 When they occur in adulthood their unique morphological and clinical features are usually not recognised. Therefore, it is recommended that immunohistochemistry for SDHB should be performed on gastric GISTs with compatible morphology. Clues to the diagnosis include a predominantly epithelioid morphology, multifocality or multinodularity and lymph node metastases. SDH deficient GISTs appear to always be positive for KIT and DOG1 by immunohistochemistry, in contrast to PDGFRA mutated gastric GISTs which may also demonstrate an epithelioid morphology but are commonly negative for KIT. We would normally perform SDHB immunohistochemistry before sequencing for KIT and PDGFRA, however an alternative approach would be to perform SDHB immunohistochemistry on all wild type GISTs because SDHB negative GISTs are invariably wild type for
SDHB positive
SDHB negative
Entire GIT (very occasionally extra-GIT) Usually (>90%) No Equal Older adult Yes Rare Spindled Rare or never Aggressive Usual Very rare Neurofibromatosis1 Germline KIT mutation Germline PDGFRA
Stomach only Never Sometimes Females more common than male Commonly child or young adult No Common Epithelioid Common Indolent Absent More common but still minority Carney triad* Carney–Stratakis/PGL syndromes{
The Carney triad is the non-hereditary association of GIST, paraganglioma and pulmonary chondroma. The molecular mechanism is unknown. Carney–Stratakis syndrome/PGL syndromes are the hereditary association of paraganglioma and GIST due to germline SDH mutations.
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SDH AND MITOCHONDRIAL DRIVEN NEOPLASIA
Fig. 3 Serially (A) H&E and (B) SDHB stained whole mount sections of a metastasis to the liver of an SDH deficient GIST. The intense staining of the mitochondria rich hepatocytes contrasts strongly with the completely negative staining of the GIST.
these two genes. Although the great majority of paediatric wild type GISTs will be SDHB negative,33 in adults it appears that only approximately one-quarter to one-third of all wild type GISTs (perhaps half of gastric wild type GISTs) will be of the SDH deficient type.21,30,32 The genetic mechanisms of neoplasia in SDH deficient GISTs is not clear in all cases. Working with a predominantly paediatric population, Janeway et al. tested for germline mutation of SDHB, SDHC and SDHD in 34 SDH deficient GISTs (without a personal or family history of paraganglioma) and found germline mutations in SDHB and SDHC in four cases (12%).33 In contrast, working with a predominantly adult population, Miettinen et al. found no germline mutations of SDHB, SDHC or SDHD in 10 cases studied.32 Because SDHA has only recently been recognised as a true tumour suppressor gene, neither of these two groups looked for SDHA mutation. However, working in a different cohort at a similar time, Pantaleo et al. have now documented SDHA mutation in four GISTs.35,36 As a significant proportion of SDH deficient GISTs are associated with SDHA, SDHB, SDHC or SDHD mutation, it is recommended that individuals with SDH deficient GISTs be offered formal genetic testing. If the immunohistochemical profile of GISTs is similar to PHEO/PGL it would be assumed that these SDHA mutated GISTs would be SDHA and
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SDHB negative by immunohistochemistry, whereas other SDH deficient GISTs would be SDHA positive but SDHB negative. Therefore, immunohistochemistry for both SDHB and SDHA may help to triage the order in which these genes are tested. However, this hypothesis has not been tested to date. GISTs arising in the setting of neurofibromatosis, although often wild type and sometimes occurring in childhood, have been consistently shown to demonstrate positive staining for SDHB, occur throughout the gastrointestinal tract and display a predominantly spindled morphology.21,32,33,37 Although they may sometimes present as paediatric wild type GISTs (or at least GISTs which arise in childhood and are wild type for KIT and PDGFRA), they are not SDH deficient GISTs. In summary the combined results of several studies indicate that germline mutation of SDHA, SDHB, SDHC or SDHD may be the cause of at least some SDH deficient GISTs and that genetic testing for each of these genes should be offered whenever an SDH deficient GIST is recognised. However, it appears that in contrast to SDHB negative paraganglioma (where the majority of SDHB negative cases will be shown to be associated with a germline mutation), the majority of SDHB negative GISTs do not harbour a germline mutation in any of the recognised components of the mitochondrial complex 2. Some of these individuals will have the Carney triad (the molecular pathogenesis of which is unknown but clearly involves dysfunction of the mitochondrial complex 2), but the majority will not. The mechanism of mitochondrial complex 2 dysfunction and tumourigenesis in these cases which are not associated with germline SDH mutation therefore is unknown. In the interim, we recommend that in addition to genetic testing for SDHA, SDHB, SDHC and SDHD, individuals diagnosed with SDH deficient GIST should be offered clinical screening and follow-up for paraganglioma and pulmonary chondroma as well as for recurrent or metachronous GIST. In addition, consideration should be given to the unique natural history of these tumours when planning treatment and follow-up.
SDHB NEGATIVE RENAL CARCINOMA Approximately 4% of renal carcinomas are hereditary and there are known associations with germline mutations of VHL, c-MET, FH and FLCN and constitutional translocations of chromosome 3.38 Germline SDHB, SDHC and SDHD mutations have now also been clearly associated with renal neoplasia.24,39 Historically these tumours have usually been classified as carcinoma (commonly conventional clear cell renal carcinoma but also chromophobe carcinoma, papillary renal carcinoma or other types) but sometimes these tumours have been classified as oncocytoma (summarised in40). In 2011
Fig. 4 Serially (A) H&E and (B) SDHB stained whole sections of a metastasis to the liver of an SDH deficient GIST. At this magnification the distinctive epithelioid morphology and somewhat nested architecture of the GIST is noted. The positive staining for SDHB in the non-neoplastic hepatocytes, endothelial and perivascular cells is again noted.
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Fig. 5 (A–D) Three of the four SDHB associated renal carcinomas we reported demonstrated similar histological features. The most striking feature and easily recognisable feature is the presence of intracytoplasmic inclusions which contain either (C) eosinophilic or (D) vacuolated material.
we demonstrated that negative staining for SDHB identifies renal neoplasia associated with germline SDHB mutation.40,41 This finding has since been confirmed by others.42 If what is known about the staining profile of PHEO/PGL can be extrapolated to renal carcinoma, it is likely that tumours associated with SDHC and SDHD mutation will also be shown to be negative for SDHB by immunohistochemistry. Indeed, recently immunohistochemistry for SDHB has been reported as being negative in one case of renal carcinoma arising in the setting of SDHC mutation.39 However, given that SDHB staining was positive in another renal carcinoma with a different morphology arising in the same patient, the hypothesis that negative staining for SDHB will also identify SDHC and SDHD mutated renal carcinomas clearly needs to be tested further before it is put into practice. It is interesting to note that in most, if not all, of the early reports of renal neoplasia arising in the setting of SDH mutation, the morphology of the tumours was not illustrated or described. When we examined a series of four renal tumours arising in the setting of germline SDHB mutation, we found that three of them were characterised by unique and distinctive morphological features (Fig. 5).40 Briefly, they were composed of cuboidal cells with bubbly eosinophilic cytoplasm and indistinct cell borders, often arranged in a nested architecture.
Many of the cells displayed distinctive cytoplasmic inclusions which were vacuolated or contained pale eosinophilic material. However, one of the tumours we encountered did not show this stereotypical morphology and demonstrated sarcomatoid and papillary areas (Fig. 6). It would have been unrecognisable if not for immunohistochemistry for SDHB. In summary, the morphological clues to SDHB associated renal neoplasia are real but subtle and clearly not present in all tumours. Therefore, immunohistochemistry for SDHB should be used to screen for SDH mutations, not only whenever these morphological clues are encountered, but also when there are clinical red flags for hereditary renal neoplasia. These clinical red flags include bilaterality, multifocality, young age of onset, or a personal or family history of either renal carcinoma or PHEO/PGL (particularly if von Hippel–Lindau syndrome has been excluded) or GIST.
OTHER TUMOURS POTENTIALLY ASSOCIATED WITH SDH DYSFUNCTION Although the link between SDHB mutation and neuroblastoma has been disputed and if such an association does occur it is clearly rare,43,44 at least three individuals with germline SDH mutation and neuroblastoma, including one who subsequently
Fig. 6 (A,B) In this example of SDHB associated renal carcinoma, the neoplastic cells are undifferentiated and there are sarcomatoid and papillary areas. (B) At higher power some very subtle cytoplasmic vacuolation (perhaps corresponding to the inclusions illustrated in Fig. 5) are present. Immunohistochemistry for SDHB was negative, illustrating the importance of performing this stain if there are clinical red flags, even if the stereotypical morphology is absent.
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SDH AND MITOCHONDRIAL DRIVEN NEOPLASIA
developed renal carcinoma, have been reported.45 Despite the rarity of the association, there may yet be a role for screening neuroblastomas for germline mutations with SDHB immunohistochemistry. A single case of an adrenal adenomatoid tumour in association with a germline SDHD mutation has been reported.46 There has also been a single growth hormone producing pituitary adenoma reported in association with SDHD mutation.47 Both these tumours were reported to show negative staining for SDHB by immunohistochemistry, supporting the argument that they are bona fide manifestations of SDHD germline mutations rather than incidental findings. However, further investigation is required before these novel associations can be considered definitive. Again immunohistochemistry for SDHB appears to be an ideal screening tool for assessing the relationship of these tumours with mitochondrial complex 2 dysfunction.
MECHANISM OF NEOPLASIA IN MITOCHONDRIAL COMPLEX 2 DYSFUNCTION The mechanisms by which mitochondrial complex 2 dysfunction causes neoplasia is not fully understood but the key events are gradually becoming apparent (reviewed in 48). Hypoxia inducible factors (HIFs) are sequence specific DNA-binding transcription factors that facilitate cell adaptation and survival in hypoxic conditions. Both mitochondrial complex deficiency and VHL mutation lead to stabilisation of HIFs. In turn the HIFs induce transcription of several genes involved in angiogenesis, energy metabolism, survival and growth. The downstream cellular events which occur in both mitochondrial complex 2 deficiency and VHL mutation therefore are very similar to those associated with hypoxia and these events have been termed a ‘pseudohypoxic response’. In contrast, the transcription profile displayed by RET, NF1, KIF1BB, TMEM127 and MAX related tumours are quite different and linked to the activation of tyrosine kinase pathways. Although the exact mechanism by which pseudohypoxia results in neoplasia is unclear, the link between PHEO/PGL tumourigenesis and cellular events mimicking hypoxia is reinforced by the remarkable but longstanding observation that individuals exposed to chronic hypoxia by living at high altitude are predisposed to paraganglioma compared to those living at sea level (summarised in 49).
CONCLUSION PHEO/PGL is a highly hereditary condition. Although only approximately 10% of patients have a positive family history, currently 30% will be associated with germline mutation and this figure may rise as more genes are described. Therefore, any PHEO/PGL should be considered potentially hereditary until this possibility is excluded. By identifying the approximately 15% of PHEO/PGL associated with mitochondrial complex 2 dysfunction, immunohistochemistry for SDHB is a vital tool for triaging genetic testing of these tumours and the yield is particularly high in extra-adrenal PHEO/PGL. The high rate of malignant behaviour of SDHB mutated PHEO/PGL is emphasised and recognition of SDHB mutation should lead to more aggressive surgery and surveillance, particularly compared to tumours arising in MEN2 and von Hippel–Lindau syndrome which are more commonly bilateral but have a low risk of metastasis. In contrast to SDHB negative renal tumours and PHEO/PGL, it appears that only a small proportion of SDH deficient GISTs
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will be associated with germline SDH mutation; others will be associated with the Carney triad but at this stage it appears that most are sporadic. The mechanism of neoplasia in these SDH deficient GISTs not associated with SDH mutation is unknown. Although we have been unable to recognise morphological differences between SDH mutated and non-mutated PHEO/ PGL, SDH deficient GISTs and a subgroup of SDHB mutated renal carcinomas show very characteristic morphological and clinical features which, if recognised, can lead to immunohistochemistry for SDHB and therefore definitive diagnosis. Early diagnosis of SDH related neoplasia is clearly advantageous in view of the distinct natural history of these tumours and the potential for genetic testing and screening to alleviate the burden of disease in these kindreds. In conclusion, after all these years it appears that Warburg was right. Some tumours (albeit relatively rare subsets of PHEO/PGL, GIST and renal carcinoma and perhaps a few other tumour types) are indeed driven primarily by mitochondrial complex 2 dysfunction. Surgical pathologists should be aware of this association and the fact that immunohistochemistry for SDHB allows definitive identification of these tumours. Conflicts of interest and sources of funding: The author declares that there is no conflict of interest. Address for correspondence: Dr A. J. Gill, Department of Anatomical Pathology, Royal North Shore Hospital, Pacific Highway, St Leonards, NSW 2065, Australia. E-mail: [email protected]
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