Outcomes of SDHB pathogenic variant carriers: A systematic review and meta-analysis.

CONTEXT: Carriers of germline pathogenic variants (PV) in succinate dehydrogenase type B (SDHB) are at increased risk of developing pheochromocytomas and paragangliomas (PPGL). Understanding their outcomes can guide recommendations for risk assessment and early detection. OBJECTIVE: We performed a systematic review and meta-analysis of the following outcomes in SDHB PV carriers: age-specific risk of developing tumors, metastatic progression, second primary tumor development, and mortality. MATERIALS AND METHODS: Pubmed, MEDLINE and EMBASE were searched. Sixteen studies met the inclusion criteria and were sorted into four outcome categories: age-specific penetrance, metastatic disease, risk of second tumour and mortality. We assessed heterogeneity and performed a meta-analysis across studies using a random effects model with the DerSimonian and Laird method. RESULTS: Penetrance of PPGL for non-proband/non-index SDHB PV carriers by age 20 was 4% (95% CI, 3%-6%), 11% (95% CI, 8%-15%) by age 40, 24% (95% CI, 19%-31%) by age 60 and 35% (95% CI, 25%-47%) by age 80. The overall risk of metastatic disease for non-proband/non-index carriers with PPGL was 9% (95% CI, 5-16%) per lifetime. In all affected cases (combining both proband/index and non-proband/non-index carriers with tumors), the risk of a second tumor was 24% (95% CI, 18-31%) and all cause 5-year mortality was 18% (95% CI 6-40%). CONCLUSION: Penetrance for PPGL in SDHB PV carriers increases linearly with age. Affected carriers are at risk of developing and dying from metastatic disease, or of developing second tumors. Lifelong surveillance is appropriate.

Distortion in transmission of pathogenic SDHB- and SDHD-mutated alleles from parent to offspring.

Phaeochromocytoma and paraganglioma are highly heritable tumours; half of those associated with a germline mutation are caused by mutations in genes for Krebs's cycle enzymes, including succinate dehydrogenase (SDH). Inheritance of SDH alleles is assumed to be Mendelian (probability of 50% from each parent). The departure from transmission of parental alleles in a ratio of 1:1 is termed transmission ratio distortion (TRD). We sought to assess whether TRD occurs in the transmission of SDHB pathogenic variants (PVs). This study was conducted with 41 families of a discovery cohort from Royal North Shore Hospital, Australia, and 41 families from a validation cohort from St. Bartholomew's Hospital, United Kingdom (UK). Inclusion criteria were a clinically diagnosed SDHB PV and a pedigree available for at least two generations. TRD was assessed in 575 participants with the exact binomial test. The transmission ratio for SDHB PV was 0.59 (P = 0.005) in the discovery cohort, 0.67 (P < 0.001) in the validation cohort, and 0.63 (P < 0.001) in the combined cohort. No parent-of-origin effect was observed. TRD remained significant after adjusting for potential confounders: 0.67 (P < 0.001) excluding families with incomplete family size data; 0.58 (P < 0.001) when probands were excluded. TRD was also evident for SDHD PVs in a cohort of 81 patients from 13 families from the UK. The reason for TRD of SDHB and SDHD PVs is unknown, but we hypothesize a survival advantage selected during early embryogenesis. The existence of TRD for SDHB and SDHD has implications for reproductive counselling, and further research into the heterozygote state.

A Clinicopathologic and Molecular Analysis of Fumarate Hydratase-deficient Pheochromocytoma and Paraganglioma.

Up to 40% of pheochromocytomas (PCCs) and paragangliomas (PGLs) are hereditary. Germline mutations/deletions in fumarate hydratase ( FH ) cause hereditary leiomyomatosis and renal cell carcinoma syndrome which manifests predominantly with FH-deficient uterine/cutaneous leiomyomas and renal cell carcinomas (RCCs)-tumors characterized by loss of immunohistochemical (IHC) expression of FH and/or positive staining for S-(2-succino)-cysteine. Occasional patients develop PCC/PGL. We investigated the incidence, morphologic, and clinical features of FH-deficient PCC/PGL. We identified 589 patients with PCC/PGLs that underwent IHC screening for FH and/or S-(2-succino)-cysteine. Eight (1.4%) PCC/PGLs were FH deficient (1.1% in an unselected population). The median age for FH-deficient cases was 55 (range: 30 to 77 y) with 50% arising in the adrenal. All 4 with biochemical data were noradrenergic. Two (25%) metastasized, 1 dying of disease after 174 months. Germline testing was performed on 7 patients, 6 of whom had FH missense mutations. None were known to have a significant family history before presentation or developed cutaneous leiomyomas, or FH-deficient RCC at extended follow-up. The patient wild-type for FH on germline testing was demonstrated to have somatic FH mutation and loss of heterozygosity corresponding to areas of subclonal FH deficiency in her tumor. One patient did not undergo germline testing, but FH mutation was demonstrated in his tumor. We conclude that FH-deficient PCC/PGL are underrecognized but can be identified by IHC. FH-deficient PCC/PGL are strongly associated with germline missense mutations but are infrequently associated with leiomyoma or RCC, suggesting there may be a genotype-phenotype correlation. FH-deficient PCC/PGL may have a higher metastatic risk.

Surveillance Improves Outcomes for Carriers of SDHB Pathogenic Variants: A Multicenter Study.

CONTEXT: Carriers of succinate dehydrogenase type B (SDHB) pathogenic variants (PVs) are at risk of pheochromocytoma and paraganglioma (PPGL) from a young age. It is widely recommended carriers enter a surveillance program to detect tumors, but there are limited studies addressing outcomes of surveillance protocols for SDHB PV carriers. OBJECTIVE: The purpose of this study was to describe surveillance-detected (s-d) tumors in SDHB PV carriers enrolled in a surveillance program and to compare their outcomes to probands. METHODS: This was a multicenter study of SDHB PV carriers with at least 1 surveillance episode (clinical, biochemical, imaging) in Australian genetics clinics. Data were collected by both retrospective and ongoing prospective follow-up. Median duration of follow-up was 6.0 years. RESULTS: 181 SDHB PV carriers (33 probands and 148 nonprobands) were assessed. Tumors were detected in 20% of nonprobands undergoing surveillance (age range 9-76 years). Estimated 10-year metastasis-free survival was 66% for probands and 84% for nonprobands with s-d tumors (P = .027). S-d tumors were smaller than those in probands (median 27 mm vs 45 mm respectively, P = .001). Tumor size ≥40 mm was associated with progression to metastatic disease (OR 16.9, 95% CI 2.3-187.9, P = .001). Patients with s-d tumors had lower mortality compared to probands: 10-year overall survival was 79% for probands and 100% for nonprobands (P = .029). CONCLUSION: SDHB carriers with s-d tumors had smaller tumors, reduced risk of metastatic disease, and lower mortality than probands. Our results suggest that SDHB PV carriers should undertake surveillance to improve clinical outcomes.

Functional significance of germline EPAS1 variants.

Mosaic or somatic EPAS1 mutations are associated with a range of phenotypes including pheochromocytoma and/or paraganglioma (PPGL), polycythemia and somatostatinoma. The pathogenic potential of germline EPAS1 variants however is not well understood. We report a number of germline EPAS1 variants occurring in patients with PPGL, including a novel variant c.739C>A (p.Arg247Ser); a previously described variant c.1121T>A (p.Phe374Tyr); several rare variants, c.581A>G (p.His194Arg), c.2353C>A (p.Pro785Thr) and c.2365A>G (p.Ile789Val); a common variant c.2296A>C (p.Thr766Pro). We performed detailed functional studies to understand their pathogenic role in PPGL. In transient transfection studies, EPAS1/HIF-2α p.Arg247Ser, p.Phe374Tyr and p.Pro785Thr were all stable in normoxia. In co-immunoprecipitation assays, only the novel variant p.Arg247Ser showed diminished interaction with pVHL. A direct interaction between HIF-2α Arg247 and pVHL was confirmed in structural models. Transactivation was assessed by means of a HRE-containing reporter gene in transiently transfected cells, and significantly higher reporter activity was only observed with EPAS1/HIF-2α p.Phe374Tyr and p.Pro785Thr. In conclusion, three germline EPAS1 variants (c.739C>A (p.Arg247Ser), c.1121T>A (p.Phe374Tyr) and c.2353C>A (p.Pro785Thr)) all have some functional features in common with somatic activating mutations. Our findings suggest that these three germline variants are hypermorphic alleles that may act as modifiers to the expression of PPGLs.

Genotype-Phenotype Features of Germline Variants of the TMEM127 Pheochromocytoma Susceptibility Gene: A 10-Year Update.

PURPOSE: This work aimed to evaluate genotype-phenotype associations in individuals carrying germline variants of transmembrane protein 127 gene (TMEM127), a poorly known gene that confers susceptibility to pheochromocytoma (PHEO) and paraganglioma (PGL). DESIGN: Data were collected from a registry of probands with TMEM127 variants, published reports, and public databases. MAIN OUTCOME ANALYSIS: Clinical, genetic, and functional associations were determined. RESULTS: The cohort comprised 110 index patients (111 variants) with a mean age of 45 years (range, 21-84 years). Females were predominant (76 vs 34, P < .001). Most patients had PHEO (n = 94; 85.5%), although PGL (n = 10; 9%) and renal cell carcinoma (RCC, n = 6; 5.4%) were also detected, either alone or in combination with PHEO. One-third of the cases had multiple tumors, and known family history was reported in 15.4%. Metastatic PHEO/PGL was rare (2.8%). Epinephrine alone, or combined with norepinephrine, accounted for 82% of the catecholamine profiles of PHEO/PGLs. Most variants (n = 63) occurred only once and 13 were recurrent (2-12 times). Although nontruncating variants were less frequent than truncating changes overall, they were predominant in non-PHEO clinical presentations (36% PHEO-only vs 69% other, P < .001) and clustered disproportionately within transmembrane regions (P < .01), underscoring the relevance of these domains for TMEM127 function. Integration of clinical and previous experimental data supported classification of variants into 4 groups based on mutation type, localization, and predicted disruption. CONCLUSIONS: Patients with TMEM127 variants often resemble sporadic nonmetastatic PHEOs. PGL and RCC may also co-occur, although their causal link requires further evaluation. We propose a new classification to predict variant pathogenicity and assist with carrier surveillance.

Bayesian approach to determining penetrance of pathogenic SDH variants.

BACKGROUND: Until recently, determining penetrance required large observational cohort studies. Data from the Exome Aggregate Consortium (ExAC) allows a Bayesian approach to calculate penetrance, in that population frequencies of pathogenic germline variants should be inversely proportional to their penetrance for disease. We tested this hypothesis using data from two cohorts for succinate dehydrogenase subunits A, B and C (SDHA-C) genetic variants associated with hereditary pheochromocytoma/paraganglioma (PC/PGL). METHODS: Two cohorts were 575 unrelated Australian subjects and 1240 unrelated UK subjects, respectively, with PC/PGL in whom genetic testing had been performed. Penetrance of pathogenic SDHA-C variants was calculated by comparing allelic frequencies in cases versus controls from ExAC (removing those variants contributed by The Cancer Genome Atlas). RESULTS: Pathogenic SDHA-C variants were identified in 106 subjects (18.4%) in cohort 1 and 317 subjects (25.6%) in cohort 2. Of 94 different pathogenic variants from both cohorts (seven in SDHA, 75 in SDHB and 12 in SDHC), 13 are reported in ExAC (two in SDHA, nine in SDHB and two in SDHC) accounting for 21% of subjects with SDHA-C variants. Combining data from both cohorts, estimated lifetime disease penetrance was 22.0% (95% CI 15.2% to 30.9%) for SDHB variants, 8.3% (95% CI 3.5% to 18.5%) for SDHC variants and 1.7% (95% CI 0.8% to 3.8%) for SDHA variants. CONCLUSION: Pathogenic variants in SDHB are more penetrant than those in SDHC and SDHA. Our findings have important implications for counselling and surveillance of subjects carrying these pathogenic variants.

TERT structural rearrangements in metastatic pheochromocytomas.

Pheochromocytomas (PC) and paragangliomas (PGL) are endocrine tumors for which the genetic and clinicopathological features of metastatic progression remain incompletely understood. As a result, the risk of metastasis from a primary tumor cannot be predicted. Early diagnosis of individuals at high risk of developing metastases is clinically important and the identification of new biomarkers that are predictive of metastatic potential is of high value. Activation of TERT has been associated with a number of malignant tumors, including PC/PGL. However, the mechanism of TERT activation in the majority of PC/PGL remains unclear. As TERT promoter mutations occur rarely in PC/PGL, we hypothesized that other mechanisms - such as structural variations - may underlie TERT activation in these tumors. From 35 PC and four PGL, we identified three primary PCs that developed metastases with elevated TERT expression, each of which lacked TERT promoter mutations and promoter DNA methylation. Using whole genome sequencing, we identified somatic structural alterations proximal to the TERT locus in two of these tumors. In both tumors, the genomic rearrangements led to the positioning of super-enhancers proximal to the TERT promoter, that are likely responsible for the activation of the normally tightly repressed TERT expression in chromaffin cells.

Analysis of SDHAF3 in familial and sporadic pheochromocytoma and paraganglioma.

BACKGROUND: Germline mutations in genes encoding subunits of succinate dehydrogenase (SDH) are associated with the development of pheochromocytoma (PC) and/or paraganglioma (PGL). As assembly factors have been identified as playing a role in maturation of individual SDH subunits and assembly of the functioning SDH complex, we hypothesized that SDHAF3 variants may be associated with PC/PGL and functionality of SDH. METHODS: DNA was extracted from the blood of 37 individuals (from 23 families) with germline SDH mutations and 18 PC/PGL (15 sporadic, 3 familial) and screened for mutations using a custom gene panel, containing SDHAF3 (SDH assembly factor 3) as well as eight known PC/PGL susceptibility genes. Molecular and functional consequences of an identified sequence variant of SDHAF3 were assessed in yeast and mammalian cells (HEK293). RESULTS: Using massively parallel sequencing, we identified a variant in SDHAF3, c.157 T > C (p.Phe53Leu), associated with increased prevalence in familial and sporadic PC/PGL (6.6%) when compared to normal populations (1.2% [1000 Genomes], p = 0.003; 2.1% [Exome Aggregation Consortium], p = 0.0063). In silico prediction tools suggest this variant is probably damaging to protein function, hence we assessed molecular and functional consequences of the resulting amino acid change (p.Phe53Leu) in yeast and human cells. We showed that introduction of SDHAF3 p.Phe53Leu into Sdh7 (ortholog of SDHAF3 in humans) null yeast resulted in impaired function, as observed by its failure to restore SDH activity when expressed in Sdh7 null yeast relative to WT SDHAF3. As SDHAF3 is involved in maturation of SDHB, we tested the functional impact of SDHAF3 c.157 T > C and various clinically relevant SDHB mutations on this interaction. Our in vitro studies in human cells show that SDHAF3 interacts with SDHB (residues 46 and 242), with impaired interaction observed in the presence of the SDHAF3 c.157 T > C variant. CONCLUSIONS: Our studies reveal novel insights into the biogenesis of SDH, uncovering a vital interaction between SDHAF3 and SDHB. We have shown that SDHAF3 interacts directly with SDHB (residue 242 being key to this interaction), and that a variant in SDHAF3 (c.157 T > C [p.Phe53Leu]) may be more prevalent in individuals with PC/PGL, and is hypomorphic via impaired interaction with SDHB.

Utility of the succinate: Fumarate ratio for assessing SDH dysfunction in different tumor types.

OBJECTIVE: Mutations of genes encoding the four subunits of succinate dehydrogenase (SDH) have been associated with pheochromocytoma and paraganglioma (PPGLs), gastrointestinal stromal tumors (GISTs) and renal cell carcinomas (RCCs). These tumors have not been characterized in a way that reflects severity of SDH dysfunction. Mass spectrometric analysis now allows measurement of metabolites extracted from formalin fixed paraffin embedded (FFPE) specimens. We assess whether SDH deficiency in various tumor types characterized by loss of SDHB protein expression correlates with SDH dysfunction as assessed by the ratio of succinate:fumarate in FFPE specimens. PATIENTS AND METHODS: Sections of FFPE tumor specimens from 18 PPGL, 10 GIST and 11 RCC patients with known SDHx mutation status for SDH deficiency were collected for mass spectrometric analysis of succinate and fumarate. RESULTS: FFPE samples showed higher succinate:fumarate ratios in SDH-deficient PPGLs compared to SDH-sufficient PPGLs. Similarly, a higher succinate:fumarate ratio was able to distinguish SDH-deficient GISTs and RCCs from their SDH-sufficient counterparts with great selectivity. Interestingly, the cut-off value of the succinate:fumarate ratio was two-folds greater in RCCs than GISTs. CONCLUSION: Analyzing biochemical imbalances preserved in FFPE specimens with mass spectrometry expands the method and sample type repertoire available for characterisation of multiple neoplasias associated with SDH deficiency.

Consensus Statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas.

Phaeochromocytomas and paragangliomas (PPGLs) are neural-crest-derived tumours of the sympathetic or parasympathetic nervous system that are often inherited and are genetically heterogeneous. Genetic testing is recommended for patients with these tumours and for family members of patients with hereditary forms of PPGLs. Due to the large number of susceptibility genes implicated in the diagnosis of inherited PPGLs, next-generation sequencing (NGS) technology is ideally suited for carrying out genetic screening of these individuals. This Consensus Statement, formulated by a study group comprised of experts in the field, proposes specific recommendations for the use of diagnostic NGS in hereditary PPGLs. In brief, the study group recommends target gene panels for screening of germ line DNA, technical adaptations to address different modes of disease transmission, orthogonal validation of NGS findings, standardized classification of variant pathogenicity and uniform reporting of the findings. The use of supplementary assays, to aid in the interpretation of the results, and sequencing of tumour DNA, for identification of somatic mutations, is encouraged. In addition, the study group launches an initiative to develop a gene-centric curated database of PPGL variants, with annual re-evaluation of variants of unknown significance by an expert group for purposes of reclassification and clinical guidance.

15 YEARS OF PARAGANGLIOMA: Clinical manifestations of paraganglioma syndromes types 1-5.

The paraganglioma (PGL) syndromes types 1-5 are autosomal dominant disorders characterized by familial predisposition to PGLs, phaeochromocytomas (PCs), renal cell cancers, gastrointestinal stromal tumours and, rarely, pituitary adenomas. Each syndrome is associated with mutation in a gene encoding a particular subunit (or assembly factor) of succinate dehydrogenase (SDHx). The clinical manifestations of these syndromes are protean: patients may present with features of catecholamine excess (including the classic triad of headache, sweating and palpitations), or with symptoms from local tumour mass, or increasingly as an incidental finding on imaging performed for some other purpose. As genetic testing for these syndromes becomes more widespread, presymptomatic diagnosis is also possible, although penetrance of disease in these syndromes is highly variable and tumour development does not clearly follow a predetermined pattern. PGL1 syndrome (SDHD) and PGL2 syndrome (SDHAF2) are notable for high frequency of multifocal tumour development and for parent-of-origin inheritance: disease is almost only ever manifest in subjects inheriting the defective allele from their father. PGL4 syndrome (SDHB) is notable for an increased risk of malignant PGL or PC. PGL3 syndrome (SDHC) and PGL5 syndrome (SDHA) are less common and appear to be associated with lower penetrance of tumour development. Although these syndromes are all associated with SDH deficiency, few genotype-phenotype relationships have yet been established, and indeed it is remarkable that such divergent phenotypes can arise from disruption of a common molecular pathway. This article reviews the clinical presentations of these syndromes, including their component tumours and underlying genetic basis.

Factors that may influence the willingness of cancer patients to consent for biobanking.

BACKGROUND: Broad or general consent given by cancer patients for their tissue, blood, and clinical information to be stored in institutional biorepositories is fundamental to enable future ethical translational cancer research. The decision to consent for biobanking will contribute to the development of advanced diagnostic and prognostic tests, as well as new therapies to improve patient outcomes. While the rate of patient participation in biobanking programs is generally reported as high worldwide, few studies have investigated factors that may influence this decision. Biobanking at our medical research institute, an associated public (government-run) university hospital, and private hospital has been established for over 20 years, with collection of certain tumor types embedded in the research culture of these institutions. In this study, we investigated factors that may influence a cancer patient's decision to give broad consent for biobanking of their specimens. METHODS: Data on patient consent were collected over a 6-month period from both government and private hospitals associated with our medical research institute. Factors considered included gender, patient age at surgery, type of malignancy (breast, duodenal, cervical, endometrial, gastric, liver, esophageal, ovarian, pancreatic, pelvic, uterine, or vulval), type of institution where surgery was performed, and timing of consent. RESULTS: Of 171 cancer patients, 159 (93%) gave broad consent for biobanking of their tissue and blood specimens for future cancer research projects receiving ethical and scientific approval. None of the factors analyzed was shown to influence a patient's decision to contribute biological specimens and clinical data to a biorepository for future medical research. CONCLUSION: Biobanking for future ethically and scientifically approved research projects in an established institution is an initiative that receives strong support from patients undergoing cancer surgery, independent of factors including gender, age, type of tumor, type of institution where surgery was performed, or timing of consent.

Succinate dehydrogenase (SDH)-deficient renal carcinoma: a morphologically distinct entity: a clinicopathologic series of 36 tumors from 27 patients.

Succinate dehydrogenase (SDH)-deficient renal carcinoma has been accepted as a provisional entity in the 2013 International Society of Urological Pathology Vancouver Classification. To further define its morphologic and clinical features, we studied a multi-institutional cohort of 36 SDH-deficient renal carcinomas from 27 patients, including 21 previously unreported cases. We estimate that 0.05% to 0.2% of all renal carcinomas are SDH deficient. Mean patient age at presentation was 37 years (range, 14 to 76 y), with a slight male predominance (M:F=1.7:1). Bilateral tumors were observed in 26% of patients. Thirty-four (94%) tumors demonstrated the previously reported morphology at least focally, which included: solid or focally cystic growth, uniform cytology with eosinophilic flocculent cytoplasm, intracytoplasmic vacuolations and inclusions, and round to oval low-grade nuclei. All 17 patients who underwent genetic testing for mutation in the SDH subunits demonstrated germline mutations (16 in SDHB and 1 in SDHC). Nine of 27 (33%) patients developed metastatic disease, 2 of them after prolonged follow-up (5.5 and 30 y). Seven of 10 patients (70%) with high-grade nuclei metastasized as did all 4 patients with coagulative necrosis. Two of 17 (12%) patients with low-grade nuclei metastasized, and both had unbiopsied contralateral tumors, which may have been the origin of the metastatic disease. In conclusion, SDH-deficient renal carcinoma is a rare and unique type of renal carcinoma, exhibiting stereotypical morphologic features in the great majority of cases and showing a strong relationship with SDH germline mutation. Although this tumor may undergo dedifferentiation and metastasize, sometimes after a prolonged delay, metastatic disease is rare in the absence of high-grade nuclear atypia or coagulative necrosis.

Succinate dehydrogenase deficiency is rare in pituitary adenomas.

Germline mutations in the succinate dehydrogenase genes (SDHA, SDHB, SDHC, and SDHD) are established as causes of pheochromocytoma/paraganglioma, renal carcinoma, and gastrointestinal stromal tumor. It has recently been suggested that pituitary adenomas may also be a component of this syndrome. We sought to determine the incidence of SDH mutation in pituitary adenomas. We performed screening immunohistochemistry for SDHB and SDHA on all available pituitary adenomas resected at our institution from 1998 to 2012. In those patients with an abnormal pattern of staining, we then performed SDH mutation analysis on DNA extracted from paraffin-embedded tissue, fresh frozen tissue, and peripheral blood. One of 309 adenomas (0.3%) demonstrated an abnormal pattern of staining, a 30 mm prolactin-producing tumor from a 62-year-old man showing loss of staining for both SDHA and SDHB. Examination of paraffin-embedded and frozen tissues confirmed double-hit inactivating somatic SDHA mutations (c.725_736del and c.989_990insTA). Neither of these mutations was present in the germline. We conclude that, although pathogenic SDH mutation may occur in pituitary adenomas and can be identified by immunohistochemistry, it appears to be a very rare event and can occur in the absence of germline mutation. SDH-deficient pituitary adenomas may be larger and more likely to produce prolactin than other pituitary adenomas. Unless suggested by family history and physical examination, it is difficult to justify screening for SDH mutations in pituitary adenomas. Surveillance programs for patients with SDH mutation may be tailored to include the possibility of pituitary neoplasia; however, this is likely to be a low-yield strategy.

Whole exome sequencing is an efficient and sensitive method for detection of germline mutations in patients with phaeochromcytomas and paragangliomas.

BACKGROUND: Genetic testing is recommended when the probability of a disease-associated germline mutation exceeds 10%. Germline mutations are found in approximately 25% of individuals with phaeochromcytoma (PCC) or paraganglioma (PGL); however, genetic heterogeneity for PCC/PGL means many genes may require sequencing. A phenotype-directed iterative approach may limit costs but may also delay diagnosis, and will not detect mutations in genes not previously associated with PCC/PGL. OBJECTIVE: To assess whether whole exome sequencing (WES) was efficient and sensitive for mutation detection in PCC/PGL. METHODS: Whole exome sequencing was performed on blinded samples from eleven individuals with PCC/PGL and known mutations. Illumina TruSeq (Illumina Inc, San Diego, CA, USA) was used for exome capture of seven samples, and NimbleGen SeqCap EZ v3.0 (Roche NimbleGen Inc, Basel, Switzerland) for five samples (one sample was repeated). Massive parallel sequencing was performed on multiplexed samples. Sequencing data were called using Genome Analysis Toolkit and annotated using annovar. Data were assessed for coding variants in RET, NF1, VHL, SDHD, SDHB, SDHC, SDHA, SDHAF2, KIF1B, TMEM127, EGLN1 and MAX. Target capture of five exome capture platforms was compared. RESULTS: Six of seven mutations were detected using Illumina TruSeq exome capture. All five mutations were detected using NimbleGen SeqCap EZ v3.0 platform, including the mutation missed using Illumina TruSeq capture. Target capture for exons in known PCC/PGL genes differs substantially between platforms. Exome sequencing was inexpensive (<$A800 per sample for reagents) and rapid (results <5 weeks from sample reception). CONCLUSION: Whole exome sequencing is sensitive, rapid and efficient for detection of PCC/PGL germline mutations. However, capture platform selection is critical to maximize sensitivity.

Familial SDHA mutation associated with pituitary adenoma and pheochromocytoma/paraganglioma.

CONTEXT: Reports of the coexistence of pituitary adenomas and pheochromocytoma/paraganglioma are uncommon. Recently germline mutations in 2 of the genes encoding succinate dehydrogenase, SDHC and SDHD, were associated with pituitary tumors. OBJECTIVE: Our aim was to determine whether the development of a pituitary adenoma was associated with SDHA mutation. PATIENTS: A 46-year-old female presented with carotid body paraganglioma (proband). Subsequently the proband's son was diagnosed with a nonfunctioning pituitary macroadenoma at age 30 years. RESULTS: An immunohistochemical analysis of the resected paraganglioma and pituitary adenoma revealed the loss of succinate dehydrogenase subunit B and succinate dehydrogenase subunit A (SDHA) expression in both tumors, with the preservation of staining in nonneoplastic tissue. Mutation analysis showed a novel SDHA mutation (c.1873C>T, p.His625Tyr) in the germline of the proband as well as in the proband's son. In the paraganglioma of the proband, in addition to the germline mutation, a somatic mutation was observed (c.1865G>A, p.Trp622*). In the pituitary adenoma of the proband's son, loss of SDHA immunoreactivity was paradoxically accompanied by loss of the mutant allele. CONCLUSIONS: This is the first report of a pituitary adenoma arising in the setting of germline SDHA mutation. The loss of SDHA protein expression in both the paraganglioma (proband) and pituitary adenoma (proband's son) argues strongly for a causative role of SDHA mutation. This report further strengthens the link between pituitary neoplasia and germline SDH mutation. Although pituitary adenomas appear rare among patients carrying SDH subunit mutations, they may have been underrecognized due to the low penetrance of disease and lack of systematic surveillance.

Loss of SDHA expression identifies SDHA mutations in succinate dehydrogenase-deficient gastrointestinal stromal tumors.

Succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GISTs) are a unique class of GIST defined by negative immunohistochemical staining for succinate dehydrogenase B (SDHB). SDH-deficient GISTs show distinctive clinical and pathologic features including absence of KIT and PDGFRA mutations, exclusive gastric location, common lymph node metastasis, a prognosis not predicted by size and mitotic rate, and indolent behavior of metastases. They may be syndromal with some being associated with the Carney Triad or germline SDHA, SDHB, SDHC, or SDHD mutations (Carney-Stratakis syndrome). It is normally recommended that genetic testing for SDHA, SDHB, SDHC, and SDHD be offered whenever an SDH-deficient GIST is encountered. However, testing for all 4 genes is burdensome and beyond the means of most centers. In this study we performed SDHA mutation and immunohistochemical analyses for SDHA on 10 SDH-deficient GISTs. Three showed negative staining for SDHA, and all of these were associated with germline SDHA mutations. In 2 tumors, 3 novel mutations were identified (p.Gln54X, p.Thr267Met, and c.1663+3G>C), none of which have previously been reported in GISTs or other SDH-associated tumors. Seven showed positive staining for SDHA and were not associated with SDHA mutation. In conclusion, 30% of SDH-deficient GISTs in this study were associated with germline SDHA mutation. Negative staining for SDHA can be used to triage formal genetic testing for SDHA when an SDH-deficient GIST is encountered.

Breast cancer-associated fibroblasts induce epithelial-to-mesenchymal transition in breast cancer cells.

Cancer-associated fibroblasts (CAFs) play a role in tumour initiation and progression, possibly by inducing epithelial-to-mesenchymal transition (EMT), a series of cellular changes that is known to underlie the process of metastasis. The aim of this study was to determine whether CAFs and surrounding normal breast fibroblasts (NBFs) are able to induce EMT markers and functional changes in breast epithelial cancer cells. Matched pairs of CAFs and NBFs were established from fresh human breast cancer specimens and characterised by assessment of CXCL12 levels, α-smooth muscle actin (α-SMA) levels and response to doxorubicin. The fibroblasts were then co-cultured with MCF7 cells. Vimentin and E-cadherin expressions were determined in co-cultured MCF7 cells by immunofluorescence and confocal microscopy as well as by western blotting and quantitative PCR. Co-cultured MCF7 cells were also assessed functionally by invasion assay. CAFs secreted higher levels of CXCL12 and expressed higher levels of α-SMA compared with NBFs. CAFs were also less sensitive to doxorubicin as evidenced by less H2AX phosphorylation and reduced apoptosis on flow cytometric analysis of Annexin V compared with NBFs. When co-cultured with MCF7 cells, there was greater vimentin and less E-cadherin expression as well as greater invasiveness in MCF7 cells co-cultured with CAFs compared with those co-cultured with NBFs. CAFs have the ability to induce a greater degree of EMT in MCF7 cell lines, indicating that CAFs contribute to a more malignant breast cancer phenotype and their role in influencing therapy resistance should therefore be considered when treating breast cancer.