Succinate Dehydrogenase B Research Articles

An immunohistochemical study of potential diagnostic and therapeutic biomarkers of wild-type gastrointestinal stromal tumours.

The aims of this study were to investigate whether succinate dehydrogenase B (SDHB), insulin growth factor 1 receptor (IGF1R), HER2, epidermal growth factor receptor (EGFR) and/or BRAF V600E immunohistochemistry could screen for wild-type gastrointestinal stromal tumours (GISTs), and to determine what proportion of wild-type GISTs expressed these proteins and might therefore represent candidates for targeted therapies. Twenty-seven wild-type GISTs and 91 KIT-mutated or PDGFRA-mutated GISTs were immunostained for SDHB, IGF1R, HER2, and EGFR. A preliminary study of the BRAF VE1 antibody showed non-specific staining, and indicated it was neither a specific nor a sensitive marker of wild-type GISTs. SDHB loss showed 100% specificity but only 37% sensitivity as such a marker. EGFR and IGF1R were expressed by 63% and 33% of the wild-type GISTs but also by 56% and 32% of the KIT/PDGFRA mutant GISTs, respectively. Therefore, adding EGFR and/or IGF1R to SDHB as a panel only decreased the specificity of SDHB loss as a marker for wild-type status. All five antibodies failed, individually or collectively, to represent highly sensitive and highly specific markers for wild-type GIST. However, whereas HER2 has been excluded as a therapeutic biomarker, both EGFR and IGF1R are expressed by some wild-type GISTs and are therefore potential therapeutic targets.

Succinate dehydrogenase B: a new prognostic biomarker in clear cell renal cell carcinoma.

Succinate dehydrogenase B (SDHB) is a mitochondrial enzyme complex subunit. Loss of SDHB protein expression has been found to correlate with SDHx gene mutations. Little is known about its expression in subtypes of renal cell carcinoma (RCC) and whether it is a prognostic indicator. Four hundred fifty renal epithelial neoplasms were analyzed for SDHB, comprising clear cell RCC (CCRCC) (n = 240), papillary RCC (n = 84), chromophobe RCC (n = 49), renal oncocytoma (n = 47), clear cell papillary RCC (CCPRCC) (n = 19), and von Hippel-Lindau (VHL)-associated CCPRCC-like tumors (n = 11). Succinate dehydrogenase B expression was graded based upon staining intensity using a 4-tiered system (0-3+), in which 3+ was strongest and complete absence was 0. Neoplasms were further categorized based upon staining extent into SDHB weak (1+-2+) and strong (3+). Succinate dehydrogenase B was strongly preserved in 131 (55%) of 240 CCRCCs, 84 (100%) of 84 papillary RCCs, 49 (100%) of 49 chromophobe RCCs, 1 (5%) of 19 CCPRCC, 5 (45%) of 11 VHL-associated CCPRCC-like tumors, and 47 (100%) of 47 renal oncocytomas. The remaining 109 CCRCCs, 18 CCPRCCs, and 6 VHL-associated CCPRCC-like tumors had weak but preserved SDHB. Succinate dehydrogenase B expression in CCRCCs with high International Society of Urological Pathology nucleolar grade (G3-G4) correlated significantly with survival (log-rank, P = .0004). Succinate dehydrogenase B is variably expressed in RCCs with clear cell morphology and strongly preserved in most other neoplasms. Therefore, weak staining, particularly in clear neoplasms, should not be misinterpreted as negative. Finally, SDHB expression in CCRCCs with high nucleolar grade (G3-G4) is significantly associated with survival, indicating it may be both a diagnostic and prognostic marker in RCC.

Selection of Reference Genes for Expression Analysis Using Quantitative Real-Time PCR in the Pea Aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae)

To facilitate gene expression study and obtain accurate qRT-PCR analysis, normalization relative to stable expressed housekeeping genes is required. In this study, expression profiles of 11 candidate reference genes, including actin (Actin), elongation factor 1 α (EF1A), TATA-box-binding protein (TATA), ribosomal protein L12 (RPL12), β-tubulin (Tubulin), NADH dehydrogenase (NADH), vacuolar-type H+-ATPase (v-ATPase), succinate dehydrogenase B (SDHB), 28S ribosomal RNA (28S), 16S ribosomal RNA (16S), and 18S ribosomal RNA (18S) from the pea aphid Acyrthosiphon pisum, under different developmental stages and temperature conditions, were investigated. A total of four analytical tools, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the suitability of these genes as endogenous controls. According to RefFinder, a web-based software tool which integrates all four above-mentioned algorithms to compare and rank the reference genes, SDHB, 16S, and NADH were the three most stable house-keeping genes under different developmental stages and temperatures. This work is intended to establish a standardized qRT-PCR protocol in pea aphid and serves as a starting point for the genomics and functional genomics research in this emerging insect model.

Succinate dehydrogenase gene mutations and DNA methylation analysis in hereditary pheochromocytoma paraganglioma syndrome

Objective To detect the genetic mutations of succinate dehydrogenase B (SDHB),SDHC,SDHD,SDHAF2 and RET,VHL in hereditary pheochromocytoma (PHEO) paraganglioma (PGL) syndrome in order to analyze the relationship between the pathogenesis and SDHx mutations and DNA methylation.Methods SDH genes,VHL and RET were analyzed for germline mutations in 97 PHEOs/PGLs patients.Correlations were analyzed between the results and the clinical characteristics including age,tumor localization,multifocality,24 h urine CA,IGF and NSE.Direct DNA sequence analysis was carried out for SDHB (1q36.1-1q35,exons1-8),SDHC (1q21,exons 1-5),SDHD (11q23,exons 1-4),SDHAF2 (11q12.2,exons 1-4),RET (10q11.2,exons10,11,13,14&15,and 16) and VHL (3p25.3,exons 1-3) genes,and promoter region methylation of SDHB was detected in PHEO/PGL peripheral blood samples.Results Germinal mutations were found in 17 patients (17.5％),with 8 cases in RET proto-oncogene (8.2％),7 cases in SDHB genes (7.2％) and 2 cases in VHL gene (2.1％).The comparison of some of the clinical features in two groups (with and without promoter region methylation of SDHB) showed significant differences (P＜0.01).Conclusions Genetic predisposition is frequent in chromaffin tissue tumors,indicating that DNA analysis is necessary.The mutation of SDHB is highly associated with abdominal PGL and the following distant metastasis (malignant PGL). Key words: Pheochromocytomas; Paragangliomas; Succinate dehydrogenase; DNA mutational analysis; DNA methylation

Can Mutations in the BAP1 Gene be Detected by Immunohisto-chemistry in Hereditary Kidney Cancers?

Background: Hereditary renal cell carcinoma (RCC) constitutes about 5% of all RCCs. The most common and well studied syndromes include, VHL, HLRCC, BHD, Familial Oncocytoma, RCC Papillary Type 1, TSC, RCC associated with Succinate dehydrogenase B (SHDB) mutations and others. Several genes, including VHL, MET, FLCN, FH and genes encoding the succinate dehydrogenase (SDH) subunits B/C/D have been identified as causative. However, the genetic basis of a significant percentage of familial RCC, some with clear cell morphology remain unknown. BAP1 (BRCA1 associated protein-1), a tumor suppressor gene that encodes a nuclear deubiquitinase, is inactivated in 15% of sporadic clear cell RCCs and its loss was associated with high tumor grade and poor prognosis. In this study, we investigated the possible role of this gene in the spectrum of RCC part of hereditary syndromes. Materials and Methods: To elucidate the role of BAP1 in all the spectrum of hereditary RCC, we studied by IHC a panel of RCCs which covers the spectrum of kidney cancers and included 10 VHL tumors, 6 HLRCCs, 8 chromophobe, 5 Hereditary Papillary Type 1, 6 Oncocytomas, 3 BHD (hybrid), and 24 sporadic clear cell RCCs. To analyze the BAP1 expression in these tumors, formalin fixed paraffin embedded (FFPE) tissues were immunostained with mouse monoclonal anti-human BAP1 antibody (Clone C-4, Santa Cruz). Results: We found that all the tumors except two showed positive nuclear staining for BAP1. The two negative cases that were negative for BAP1 were Clear cell type and belonged to two siblings. Molecular analysis in a prepublished study showed both patients harboring the p.L14H mutation. Conclusion: Our study supports the hypothesis that BAP1 mutations can play a role in hereditary syndromes predominantly in clear cell tumors. Staining for BAP1 should be done when there is no definite known mutation in a clear cell cancer but the patient gives history of familial kidney cancer. The two related patients who had similar mutations had aggressive, metastatic disease, which suggests that probably BAP1 does play a role in hereditary RCC clear cell type.

SDHB mutations are associated with response to temozolomide in patients with metastatic pheochromocytoma or paraganglioma.

Cyclophosphamide-dacarbazine-vincristine regimen is recommended for the treatment of malignant pheochromocytoma and paraganglioma (MPP); however, dacarbazine is the only recognized active drug in neuroendocrine tumours. We investigated the therapeutic benefit of temozolomide (TMZ), an oral alternative to dacarbazine, in patients with MPP. This is a retrospective study of consecutive patients with documented progressive MPP. We examined the correlation between Succinate dehydrogenase B (SDHB) mutation and O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation and MGMT expression in the French nation-wide independent cohort of 190 pheochromocytomas or paragangliomas (PP). Progression-free survival (PFS) according to RECIST 1.1 and PERCIST 1.0 criteria was the primary end point. Fifteen consecutive patients with MPP were enrolled; ten (67%) carried a mutation in SDHB. The mean dose intensity of TMZ was 172 mg/m(2) /d for 5 days every 28 days. Median PFS was 13.3 months after a median follow-up of 35 months. There were five partial responses (33%), seven stable (47%) and three progressive diseases (20%). Grade 3 toxicities were lymphopenia in two patients and hypertension in one. Partial responses were observed only in patients with mutation in SDHB. MGMT immunohistochemistry was negative in tumour samples from four patients who responded to treatment. SDHB germline mutation was associated with hypermethylation of the MGMT promoter and low expression of MGMT in 190 samples of the French nation-wide independent cohort. This study demonstrates that TMZ is an effective antitumour agent in patients with SDHB-related MPP. The silencing of MGMT expression as a consequence of MGMT promoter hypermethylation in SDHB-mutated tumours may explain this finding.

Usefulness of Succinate dehydrogenase B (SDHB) immunohistochemistry in guiding mutational screening among patients with pheochromocytoma-paraganglioma syndromes.

Genetic testing of pheochromocytomas (PCC) and paragangliomas (PGL), although expensive, is gradually becoming a part of the routine laboratory investigation for patients with PCC-PGL syndrome. Recently, Succinate dehydrogenase B (SDHB) immunochemistry has been shown to be an excellent indicator of germline mutations in the SDH genes and could help significantly reduce cost. This study assesses the utility of SDHB immunohistochemical analysis when used to guide genetic analysis, with emphasis on cost benefits it could provide in a resource-limited setting. Forty-four cases of PCC/PGL characterized by genetic analysis were included to determine their SDHB expression pattern by immunohistochemistry. SDHB antibody expression was negative among three cases each, with SDHB and SDHD mutations. Immunohistochemistry results were positive for all three cases of RET, a single case of neurofibromatosis and for two cases with Von Hippel-Lindau (VHL) mutations while the remaining two cases with VHL mutations showed a diffuse 'cytoplasmic blush'. Thirty of the remaining 31 samples demonstrated positive staining and were negative for mutations, while a lone sample that was negative for staining and mutation was not included in the final analysis as the internal control for the sample was not adequately stained. Cost analysis in our settings showed that triaging with SDHB immunohistochemistry could potentially reduce costs by USD 320-500 per patient. SDHB immunohistochemistry, when used as a guide to genetic testing, can significantly reduce the effort, time and costs of testing among patients with PCC-PGL, a huge benefit in resource limited settings.

Classification and Proposed Nomenclature for Inherited Defects of Thyroid Hormone Action, Cell Transport, and Metabolism

Resistance to thyroid hormone (RTH) was first described in 1967 (1), and the first mutations in the THRB gene were identified in 1989 (2,3), only three years after the cloning of the THR genes (4,5). The cardinal features of this syndrome of reduced sensitivity to thyroid hormone are elevated serum levels of free thyroid hormone with nonsuppressed thyrotropin (TSH), often with goiter and no clear symptoms and signs of thyrotoxicosis (6). In fact, signs of decreased and increased thyroid hormone action in different tissues may coexist. During the First International Workshop on Resistance to Thyroid Hormone in Cambridge, United Kingdom, in 1993, a consensus statement was issued to establish a unified nomenclature of THRB gene mutations in RTH (7), as defined above. In the ensuing years more than 3000 cases have been identified, 80% of which harbored mutations in the THRB gene. More recently, two syndromes with reduced cellular access of the biologically active thyroid hormone, triiodothyronine (T3), were identified. These are caused by defects of thyroid hormone cell membrane transport (8,9) and a defect reducing the intracellular metabolism generating T3 from thyroxine (T4) (10). To accommodate these new findings, it was proposed to broaden the definition of hormone resistance. Thus, the Fifth International Workshop on Resistance to Thyroid Hormone, which took place in Lyon, France, in 2005, saw the introduction of the term “reduced sensitivity to thyroid hormone (RSTH) to encompass all defects that can interfere with the biological activity of a chemically intact thyroid hormone secreted in normal or excessive amounts.” Following the 10th International Workshop on Resistance to Thyroid Hormone and Action that took place in Quebec City, Canada, in 2012, a number of investigators took on the task to develop a nomenclature for inherited forms of impaired sensitivity to thyroid hormone (Table 1). The term “impaired” was to substitute for “reduced” because nascent data indicate that syndromes of increased sensitivity may also exist. We are cognizant that no nomenclature can fit perfectly all aspects of the described syndromes because variability exists. Several aspects were taken into consideration: the already existing nomenclature, new findings, and anticipated putative discoveries. For example, in over 2000 publications “RTH” is used to define a phenotype of congenitally increased free T4 with nonsuppressed TSH, irrespective of the presence or absence of a THRB gene mutation (see non-TR-RTH). In view of the identification of THRA gene mutations that present a distinct phenotype (11,12), we propose using the term “RTH α,” and in new publications to use “RTH β” when a THRB gene mutation is present in association with the RTH phenotype. This allows the naming of new gene defects in individuals with the RTH phenotype. The use of the abbreviation “THR” as a synonym for RTH is discouraged, not only because the hormone is not resistant, but also because this abbreviation is used to denote other circumstances. Indeed, a Medline search using THR yielded over 20,000 references, only a few related to resistance to thyroid hormone. Table 1. Inheritable Forms of Impaired Sensitivity to Thyroid Hormone

Wild-type GIST: a comprehensive review

34 Wild-type GIST: a comprehensive review 1 3 Abstract Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. The vast majority demonstrates mutually exclusive KIT or PDGFRA gain-of-function mutations. KIT or PDGFRA mutation is an early event in GIST pathogenesis and development, and has led to the introduction of KIT and PDGFRA kinase inhibitors in GIST, which has tremendously changed survival of patients suffering from this disease. Approximately 85 % of pediatric GISTs and 10–15 % of adult GISTs are devoid of mutations in the KIT and PDGFRA genes and are referred to as wild-type GISTs (WT-GIST). Over the past years, it has been shown that WT-GISTs are profoundly different from mutant GIST with regard to their clinical behavior as well as their molecular profile, and are now considered a separate pathological entity. Based on their significant molecular and clinical heterogeneity, WT-GISTs should be considered a group of diseases rather than a single disease entity. The following short review will exclusively focus on WT-GISTs, a group of tumors traditionally divided into a syndromic and a nonsyndromic (sporadic) subgroup. In addition, immunohistochemical expression of succinate dehydrogenase B (SDHB) has recently been introduced to subtype WT-GISTs into an SDHB-positive (proficient) from SDHB-negative (deficient) group.

Estrogen receptor β activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo

Estrogen receptor (ER)-β has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERβ coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERβ, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERβ agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERβ-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Landscape of the mitochondrial Hsp90 metabolome in tumours

Reprogramming of tumor cell metabolism contributes to disease progression and resistance to therapy, but how this process is regulated on the molecular level is unclear. Here we report that Heat Shock Protein 90 (Hsp90)-directed protein folding in mitochondria controls central metabolic networks in tumor cells, including the electron transport chain, citric acid cycle, fatty acid oxidation, amino acid synthesis, and cellular redox status. Specifically, mitochondrial Hsp90, but not cytosolic Hsp90, binds and stabilizes the electron transport chain Complex II subunit succinate dehydrogenase-B, maintaining cellular respiration under low-nutrient conditions, and contributing to hypoxia-inducible factor-1α-mediated tumorigenesis in patients carrying succinate dehydrogenase-B mutations. Thus, Hsp90-directed proteostasis in mitochondria regulates tumor cell metabolism, and may provide a tractable target for cancer therapy.

Paragangliomas of the urinary bladder: Experience at the National Cancer Institute.

307 Background: Bladder paragangliomas (BP, also referred to as bladder pheochromocytomas) represent 0.06% of bladder tumors. They occur both sporadically and with familial cancer syndromes such as von Hippel-Lindau (VHL) or Succinate Dehydrogenase-B (SDH-B) deficiency. Here we describe the clinical manifestations and management of BP at our institution. Methods: A retrospective review was performed of all cases of BP treated by the Urologic Oncology Branch at the National Institutes of Health (NIH) Clinical Center from 1989 – 2012. Using an approved database, we reviewed the demographics, radiologic, pathologic, laboratory data, surgical approach, and clinical outcome of patients with BP. Results: A total of 7 patients were treated for BP. Five were part of familial syndromes (3 with VHL, 2 with SDH-B deficiency, mean tumor volume (TV): 4cc), while 2 were sporadic cases (mean TV: 43cc). There was no significant age or gender difference in sporadic versus familial patients. 6 of the 7 patients presented with at least two of the three classic symptoms of headache, palpitations, and diaphoresis. 3 patients had micturition-related symptoms. For diagnosis, urine and plasma normetanephrines and urine norephinephrine showed the greatest sensitivity (0.75, 0.75, and 0.8, respectively). MRI was performed in all patients and had a sensitivity of 100% for detection of the mass. MIBG scan was also 100% sensitive, but was only performed in 3 of the 7 patients. CT missed the lesion in one patient. 3 patients were treated with TURBT (mean TV: 2.2cc), 2 underwent partial cystectomies (mean TV: 11.6cc), and one had a radical cystectomy (TV: 63cc). 2 patients had metastatic disease detected 7 and 9 years after initial treatment. No difference was seen in outcome based on the treatment modality. Conclusions: BP in familial and sporadic cases present with similar characteristics except for size. BP is best detected with catecholamines and MIBG/MRI. Surgical management is largely dictated by the extent of disease and includes TURBT, partial, and radical cystectomy. Long-term monitoring is recommended as distant recurrences can occur.

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.

Update on renal tumours

This presentation will give an update on renal tumours encompassing new entities [including tubulocystic renal cell carcinoma (RCC), acquired cystic disease associated RCC, clear cell tubulopapillary RCC, t(6;11) translocation carcinoma, and hereditary leiomyomatosis and RCC] and emerging entities [thyroid-like follicular RCC, succinate dehydrogenase B (SDHB) mutation associated RCC, and ALK-translocation associated RCC]. A summary of the entities accepted in these categories by consensus at the International Society of Urological Pathology meeting in Vancouver in March 2012 will be given. Discussion of important familial tumours involving the kidney will be included.

Warburg Effect’s Manifestation in Aggressive Pheochromocytomas and Paragangliomas: Insights from a Mouse Cell Model Applied to Human Tumor Tissue

A glycolytic profile unifies a group of pheochromocytomas and paragangliomas (PHEOs/PGLs) with distinct underlying gene defects, including von Hippel-Lindau (VHL) and succinate dehydrogenase B (SDHB) mutations. Nevertheless, their tumor aggressiveness is distinct: PHEOs/PGLs metastasize rarely in VHL-, but frequently in SDHB-patients. To date, the molecular mechanisms causing the more aggressive phenotype in SDHB-PHEOs/PGLs remain largely unknown. Recently, however, an excellent model to study aggressive PHEOs (mouse tumor tissue (MTT) cells) has been developed from mouse PHEO cells (MPC). We employed this model for a proteomics based approach to identify changes characteristic for tumor aggressiveness, which we then explored in a homogeneous set of human SDHB- and VHL-PHEOs/PGLs. The increase of glucose transporter 1 in VHL, and of hexokinase 2 in VHL and SDHB, confirmed their glycolytic profile. In agreement with the cell model and in support of decoupling of glycolysis, the Krebs cycle and oxidative phosphorylation (OXPHOS), SDHB tumors showed increased lactate dehydrogenase levels. In SDHB-PGLs OXPHOS complex activity was increased at complex III and, as expected, decreased at complex II. Moreover, protein and mRNA expression of all tested OXPHOS-related genes were higher in SDHB- than in VHL-derived tumors. Although there was no direct evidence for increased reactive oxygen species production, elevated superoxide dismutase 2 expression may reflect elevated oxidative stress in SDHB-derived PHEOs/PGLs. For the first time, we show that despite dysfunction in complex II and evidence for a glycolytic phenotype, the Warburg effect does not seem to fully apply to SDHB-PHEOs/PGLs with respect to decreased OXPHOS. In addition, we present evidence for increased LDHA and SOD2 expression in SDHB-PHEOs/PGLs, proteins that have been proposed as promising therapeutic targets in other cancers. This study provides new insight into pathogenic mechanisms in aggressive human PHEOs/PGLs, which may lead to identifying new diagnostic and prognostic markers in the near future.

Short time incubation at low temperature for retrieval of some antigens from formalin-fixed and paraffin-embedded tissues

Antibody manufacturers often recommend antigen retrieval for formalin-fixed paraffin-embedded tissue sections before immunohistochemical staining. The most commonly used methods involve high temperatures (90–120°C) in buffers. Low-temperature retrieval methods allow the use of tissue flotation baths but their incubation times are often long. This study investigated retrieval of some antigens at low temperature (65°C) for 10 minutes in citrate buffer (pH 6·0) using a tissue flotation bath. Sections were obtained from two tissue specimens, breast and O. volvulus nodules, which were formalin-fixed and paraffin-embedded. The antigens of lactate dehydrogenase, pyruvate dehydrogenase kinase-2, malic enzyme-1, succinate dehydrogenase-B, glucose-6-phosphate dehydrogenase, alpha/beta-hydrolase-4, estrogen receptors (ER), and progesterone receptors (PR) were retrieved at low temperature and in the cases of ER and PR, were also retrieved at high temperature (120°C), using a pressure cooker, for comparison. All the retrievals were carried out in citrate buffer, pH 6·0. The results showed that all the enzymes scored 2+ (i.e. a strong reaction) in staining intensity and the staining for ER and PR were similar for both methods (2+). In immunohistochemical staining, the most important stage is antigen retrieval. The low-temperature antigen retrieval in citrate buffer at pH 6·0 provided staining intensities comparable to those by high-temperature methods. The results indicate that low-temperature antigen retrieval is cost-effective, rapid, and reliable.

Use of SDHB immunohistochemistry to identify germline mutations of SDH genes

Pheochromocytomas and paragangliomas occur sporadically but are commonly associated with the von Hippel Lindau (VHL) syndrome, multiple endocrine neoplasia type 2 (MEN2), neurofibromatosis type 1 (NF1) and germline mutations of succinate dehydrogenase B (SDHB), C (SDHC) or D (SDHD). It is therefore recommended that genetic testing be considered if not performed in all cases of even apparently sporadic pheochromocytomas or paragangliomas. Recently it has been demonstrated that immunohistochemistry (IHC) for SDHB is negative in all SDH mutated paragangliomas regardless of whether the B,C or D subunit is involved [1,2]. Furthermore some clearly syndromic paragangliomas without known genetic mutation (including but not limited to those which occur in the Carney Triad) are identified by negative staining for SDHB [3]. Although historically the renal tumours occurring in the setting of SDHB mutation were usually classified as conventional clear cell carcinoma or oncocytoma, they actually display a unique morphology (unrecognized until know) and which can be confirmed by immunohistochemistry. The GISTs occurring in SDH mutation and Carney Triad are also unique and demonstrate quite a different morphology, natural history and molecular pathogenesis compared to other GISTs occurring in adults (but similar to most GISTs occurring in childhood). We call this unique subtype of GIST the type 2 GIST. Briefly type 2 GISTs arise in the stomach, show an epithelioid morphology, are often multifocal, commonly show lymph node metastasis, are wild type for KIT and PDGFR, have a prognosis not predicted by size and mitotic rate, never respond to imatinib but demonstrate an indolent growth despite the presence of frequent metastases [3,5]. We recommend that all paragangliomas, GISTs which potentially display type 2 morphological or clinical features and renal carcinomas which display the unique morphology we described should undergo immunohistochemistry for SDHB. Negative staining for SDHB indicates an abnormality of the mitochondrial complex 2 and is an absolute indication for formal genetic testing. We perform and interpret SDHB immunohistochemistry of archived formalin fixed paraffin embedded tissue in a manner analogous to MSI testing in colon cancer. In the setting of paraganglioma or renal carcinoma negative staining almost always indicates germline SDHB,SDHC or SDHD mutation (greater than 90% chance) but may indicate Carney Triad. In the setting of GIST, Carney Triad is more likely, but SDHB, SDHC or SDHD mutation accounts for at least 25% of type 2 GIST. Table 1 Syndromes associated with paraganglioma and pheochromocytoma The mitochondrial complex 2 links the Krebs cycle and the electron transport chain and is illustrated below: Figure 1

A Catecholamine-Secreting Skull Base Sinonasal Paraganglioma Presenting with Labile Hypertension in a Patient with Previously Undiagnosed Genetic Mutation

Sinonasal paragangliomas are very uncommon neuroendocrine tumors that can present as skull base lesions. Functional paragangliomas are exceedingly rare. They can be associated with genetic mutations that have been associated with increased risk of head and neck paragangliomas. We present a case of a rare functioning sinonasal paraganglioma of the skull base in a patient with distant history of prior abdominal paragangliomas. The patient underwent subtotal endoscopic resection of the skull base lesion limited by carotid encasement of the tumor. They were treated with postoperative adjuvant radiation and therapeutic metaiodobenzylguanidine (MIBG) therapy. Genetic testing revealed succinate dehydrogenase B (SDHB) mutation. Skull base paragangliomas are rare tumors that may preclude complete surgical resection. 131Iodine-MIBG can be used as adjuvant therapy in postoperative external beam radiation and in MIBG avid tumors. Long-term follow-up is needed given locally aggressive nature of these tumors, especially for patients with history of genetic mutations such as SDHB mutations as recurrent paragangliomas may develop.

Renal Tumors Associated With Germline SDHB Mutation Show Distinctive Morphology

Germline succinate dehydrogenase B (SDHB) mutation causes pheochromocytoma/paraganglioma syndrome type 4 (PGL4). PGL4 is characterized by pheochromocytoma and paraganglioma, type 2 (SDHB negative) gastrointestinal stromal tumors and renal tumors, which are usually classified as carcinoma. We report 4 kindreds with 5 PGL4-associated renal tumors. Four of the tumors occurred before the age of 30 years, 4 were in the left kidney, 3 were in female patients, and 4 demonstrated consistent but previously unrecognized morphology. The tumors were composed of cuboidal cells with bubbly eosinophilic cytoplasm and indistinct cell borders. Many of the cells displayed distinctive cytoplasmic inclusions, which were vacuolated or contained eosinophilic fluid-like material. The cells were arranged in solid nests or in tubules surrounding central spaces. The tumors were well circumscribed or lobulated and frequently showed cystic change. Benign tubules or glomeruli were often entrapped at the edges of the tumors. The fifth tumor lacked these features but displayed sarcomatoid dedifferentiation. Immunohistochemistry for SDHB was completely negative in all 4 available tumors. Death from metastatic disease occurred in the patient with dedifferentiated tumor 1 year after diagnosis, whereas the other 4 tumors were cured by local excision alone (mean follow-up, 11 y; range, 2 to 30 y). We conclude that morphology supported by negative immunohistochemistry for SDHB can be used to identify kindreds with germline SDHB mutations (PGL4 syndrome) presenting with this unique type of renal tumor. These renal tumors appear to have a good prognosis after complete excision unless there is sarcomatoid dedifferentiation.

Multiple Paragangliomas in a Pregnant Patient with a Succinate Dehydrogenase B Mutation

In this case report, we document the clinical course, laboratory findings, and imaging findings of a 21-year-old pregnant woman with multiple paragangliomas due to a succinate dehydrogenase B (SDHB) mutation. We also review the literature on previously reported cases. The patient presented with nausea, palpitations, angina, and amenorrhea. Her blood pressure was 170/100 mm Hg and her beta-human chorionic gonadotropin was positive. Her blood pressure remained high despite phenoxybenzamine titration. A 24-hour urine analysis revealed elevated plasma metanephrines. Imaging was initially deferred due to early gestational age. After the patient terminated the pregnancy, magnetic resonance imaging revealed a left suprarenal mass, a mass at the aortic bifurcation, and a left periaortic mass. Her blood pressure was controlled on phenoxybenzamine and labetalol. The masses were excised. Pathology revealed paragangliomas. Genetic testing revealed mutation in the SDHB gene. One month later, her blood pressure was 122/86 mm Hg off of medication. Paraganglioma/pheochromocytoma should be suspected when hypertension occurs early in pregnancy. Genetic testing is important, as this may impact future offspring.