Maya Lodish

Defects in succinate dehydrogenase in gastrointestinal stromal tumors lacking KIT and PDGFRA mutations

Carney-Stratakis syndrome, an inherited condition predisposing affected individuals to gastrointestinal stromal tumor (GIST) and paraganglioma, is caused by germline mutations in succinate dehydrogenase (SDH) subunits B, C, or D, leading to dysfunction of complex II of the electron transport chain. We evaluated the role of defective cellular respiration in sporadic GIST lacking mutations in KIT or PDGFRA (WT). Thirty-four patients with WT GIST without a personal or family history of paraganglioma were tested for SDH germline mutations. WT GISTs lacking demonstrable SDH genetic inactivation were evaluated for SDHB expression by immunohistochemistry and Western blotting and for complex II activity. For comparison, SDHB expression was also determined in KIT mutant and neurofibromatosis-1–associated GIST, and complex II activity was also measured in SDH-deficient paraganglioma and KIT mutant GIST; 4 of 34 patients (12%) with WT GIST without a personal or family history of paraganglioma had germline mutations in SDHB or SDHC. WT GISTs lacking somatic mutations or deletions in SDH subunits had either complete loss of or substantial reduction in SDHB protein expression, whereas most KIT mutant GISTs had strong SDHB expression. Complex II activity was substantially decreased in WT GISTs. WT GISTs, particularly those in younger patients, have defects in SDH mitochondrial complex II, and in a subset of these patients, GIST seems to arise from germline-inactivating SDH mutations. Testing for germline mutations in SDH is recommended in patients with WT GIST. These findings highlight a potential central role of SDH dysregulation in WT GIST oncogenesis.

SDHB immunohistochemistry: a useful tool in the diagnosis of Carney–Stratakis and Carney triad gastrointestinal stromal tumors

Mutations in the tumor suppressor genes SDHB, SDHC, and SDHD (or collectively SDHx) cause the inherited paraganglioma syndromes, characterized by pheochromocytomas and paragangliomas. However, other tumors have been associated with SDHx mutations, such as gastrointestinal stromal tumors (GISTs) specifically in the context of Carney–Stratakis syndrome. Previously, we have shown that SDHB immunohistochemistry is a reliable technique for the identification of pheochromocytomas and paragangliomas caused by SDHx mutations. We hypothesized that GISTs in patients with SDHx mutations would be negative immunohistochemically for SDHB as well. Four GISTs from patients with Carney–Stratakis syndrome and six from patients with Carney triad were investigated by SDHB immunohistochemistry. Five GISTs with KIT or PDGFRA gene mutations were used as controls. In addition, SDHB immunohistochemistry was performed on 42 apparently sporadic GISTs. In cases in which the SDHB immunohistochemistry was negative, mutational analysis of SDHB, SDHC, and SDHD was performed. All GISTs from patients with Carney–Stratakis syndrome and Carney triad were negative for SDHB immunohistochemically. In one patient with Carney–Stratakis syndrome, a germline SDHB mutation was found (p.Ser92Thr). The five GISTs with a KIT or PDGFRA gene mutation were all immunohistochemically positive for SDHB. Of the 42 sporadic tumors, one GIST was SDHB-negative. Mutational analysis of this tumor did not reveal an SDHx mutation. All SDHB-negative GISTs were located in the stomach, had an epithelioid morphology, and had no KIT or PDGFRA mutations. We show that Carney–Stratakis syndrome- and Carney-triad-associated GISTs are negative by immunohistochemistry for SDHB in contrast to KIT- or PDGFRA-mutated GISTs and a majority of sporadic GISTs. We suggest that GISTs of epithelioid cell morphology are tested for SDHB immunohistochemically. In case of negative SDHB staining in GISTs, Carney–Stratakis syndrome or Carney triad should be considered and appropriate clinical surveillance should be instituted.

The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes.

Stratakis CA, Tichomirowa MA, Boikos S, Azevedo MF, Lodish M, Martari M, Verma S, Daly AF, Raygada M, Keil MF, Papademetriou J, Drori‐Herishanu L, Horvath A, Tsang KM, Nesterova M, Franklin S, Vanbellinghen J‐F, Bours V, Salvatori R, Beckers A. The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes.

Metastatic Pheochromocytoma/Paraganglioma Related to Primary Tumor Development in Childhood or Adolescence: Significant Link to SDHB Mutations

PURPOSE To present data on the high rate of SDHB mutations in patients with metastatic pheochromocytoma/paraganglioma whose initial tumor presentation began in childhood or adolescence. PATIENTS AND METHODS From 2000 to 2010, 263 patients with pheochromocytoma/paraganglioma were evaluated through the National Institutes of Health (NIH), Bethesda, MD. Of the 263 patients, 125 patients were found to have metastatic disease; of these 125 patients, 32 patients presented with a tumor before 20 years of age. An additional 17 patients presented with a tumor before 20 years of age but demonstrated no development of metastatic disease. Genetic testing for mutations in the VHL, MEN, and SDHB/C/D genes was performed on patients without previously identified genetic mutations. RESULTS Of the 32 patients who presented with metastatic disease and had their primary tumor in childhood or adolescence, sequence analysis of germline DNA showed SDHB mutations in 23 patients (71.9%), SDHD mutations in three patients (9.4%), VHL mutations in two patients (6.3%), and an absence of a known mutation in four patients (12.5%). The majority of these 32 patients (78.1%) presented with primary tumors in an extra-adrenal location. CONCLUSION The majority of patients with metastatic pheochromocytoma/paraganglioma who presented with a primary tumor in childhood/adolescence had primary extra-adrenal tumors and harbored SDHB mutations. Except for primary tumors located in the head and neck where SDHD genetic testing is advised, we recommend that patients who present with metastatic pheochromocytoma/paraganglioma with primary tumor development in childhood or adolescence undergo SDHB genetic testing before they undergo testing for other gene mutations, unless clinical presentation or family history suggests a different mutation.

Vandetanib in Children and Adolescents with Multiple Endocrine Neoplasia Type 2B Associated Medullary Thyroid Carcinoma

Purpose: Medullary thyroid carcinoma (MTC) is a manifestation of multiple endocrine neoplasia type 2 (MEN2) syndromes caused by germline, activating mutations in the RET (REarranged during Transfection) proto-oncogene. Vandetanib, a VEGF and EGF receptor inhibitor, blocks RET tyrosine kinase activity and is active in adults with hereditary MTC. Experimental Design: We conducted a phase I/II trial of vandetanib for children (5–12 years) and adolescents (13–18 years) with MTC to define a recommended dose and assess antitumor activity. The starting dose was 100 mg/m2 administered orally, once daily, continuously for 28-day treatment cycles. The dose could be escalated to 150 mg/m2/d after two cycles. Radiographic response to vandetanib was quantified using RECIST (v1.0), biomarker response was measured by comparing posttreatment serum calcitonin and carcinoembryonic antigen (CEA) levels to baseline, and a patient-reported outcome was used to assess clinical benefit. Results: Sixteen patients with locally advanced or metastatic MTC received vandetanib for a median (range) 27 (2–52) cycles. Eleven patients remain on protocol therapy. Diarrhea was the primary dose-limiting toxicity. In subjects with M918T RET germline mutations (n = 15) the confirmed objective partial response rate was 47% (exact 95% confidence intervals, 21%–75%). Biomarker partial response was confirmed for calcitonin in 12 subjects and for CEA in 8 subjects. Conclusion: Using an innovative trial design and selecting patients based on target gene expression, we conclude that vandetanib 100 mg/m2/d is a well-tolerated and highly active new treatment for children and adolescents with MEN2B and locally advanced or metastatic MTC. Clin Cancer Res; 19(15); 4239–48. ©2013 AACR.

RET oncogene in MEN2, MEN2B, MTC and other forms of thyroid cancer

Hereditary medullary thyroid carcinoma (MTC) is caused by specific autosomal dominant gain-of-function mutations in the RET proto-oncogene. Genotype–phenotype correlations exist that help predict the presence of other associated endocrine neoplasms as well as the timing of thyroid cancer development. MTC represents a promising model for targeted cancer therapy, as the oncogenic event responsible for initiating malignancy has been well characterized. The RET proto-oncogene has become the target for molecularly designed drug therapy. Tyrosine kinase inhibitors targeting activated RET are currently in clinical trials for the treatment of patients with MTC. This review will provide a brief overview of MTC and the associated RET oncogenic mutations, and will summarize the therapies designed to strategically interfere with the pathologic activation of the RET oncogene.

Frequent Phosphodiesterase 11A Gene (PDE11A) Defects in Patients with Carney Complex (CNC) Caused by PRKAR1A Mutations: PDE11A May Contribute to Adrenal and Testicular Tumors in CNC as a Modifier of the Phenotype

BACKGROUND Carney complex (CNC) is an autosomal dominant multiple neoplasia, caused mostly by inactivating mutations of the regulatory subunit 1A of the protein kinase A (PRKAR1A). Primary pigmented nodular adrenocortical disease (PPNAD) is the most frequent endocrine manifestation of CNC with a great inter-individual variability. Germline, protein-truncating mutations of phosphodiesterase type 11A (PDE11A) have been described to predispose to a variety of endocrine tumors, including adrenal and testicular tumors. OBJECTIVES Our objective was to investigate the role of PDE11A as a possible gene modifier of the phenotype in a series of 150 patients with CNC. RESULTS A higher frequency of PDE11A variants in patients with CNC compared with healthy controls was found (25.3 vs. 6.8%, P < 0.0001). Among CNC patients, those with PPNAD were significantly more frequently carriers of PDE11A variants compared with patients without PPNAD (30.8 vs. 13%, P = 0.025). Furthermore, men with PPNAD were significantly more frequently carriers of PDE11A sequence variants (40.7%) than women with PPNAD (27.3%) (P < 0.001). A higher frequency of PDE11A sequence variants was also found in patients with large-cell calcifying Sertoli cell tumors (LCCSCT) compared with those without LCCSCT (50 vs. 10%, P = 0.0056). PDE11A variants were significantly associated with the copresence of PPNAD and LCCSCT in men: 81 vs. 20%, P < 0.004). The simultaneous inactivation of PRKAR1A and PDE11A by small inhibitory RNA led to an increase in cAMP-regulatory element-mediated transcriptional activity under basal conditions and after stimulation by forskolin. CONCLUSIONS We demonstrate, in a large cohort of CNC patients, a high frequency of PDE11A variants, suggesting that PDE11A is a genetic modifying factor for the development of testicular and adrenal tumors in patients with germline PRKAR1A mutation.

Macronodular Adrenal Hyperplasia due to Mutations in an Armadillo Repeat Containing 5 (ARMC5) Gene: A Clinical and Genetic Investigation

CONTEXT Inactivating germline mutations of the probable tumor suppressor gene, armadillo repeat containing 5 (ARMC5), have recently been identified as a genetic cause of macronodular adrenal hyperplasia (MAH). OBJECTIVE We searched for ARMC5 mutations in a large cohort of patients with MAH. The clinical phenotype of patients with and without ARMC5 mutations was compared. METHODS Blood DNA from 34 MAH patients was genotyped using Sanger sequencing. Diurnal serum cortisol measurements, plasma ACTH levels, urinary steroids, 6-day Liddles test, adrenal computed tomography, and weight of adrenal glands at adrenalectomy were assessed. RESULTS Germline ARMC5 mutations were found in 15 of 34 patients (44.1%). In silico analysis of the mutations indicated that seven (20.6%) predicted major implications for gene function. Late-night cortisol levels were higher in patients with ARMC5-damaging mutations compared with those without and/or with nonpathogenic mutations (14.5 ± 5.6 vs 6.7 ± 4.3, P < .001). All patients carrying a pathogenic ARMC5 mutation had clinical Cushings syndrome (seven of seven, 100%) compared with 14 of 27 (52%) of those without or with mutations that were predicted to be benign (P = .029). Repeated-measures analysis showed overall higher urinary 17-hydroxycorticosteroids and free cortisol values in the patients with ARMC5-damaging mutations during the 6-day Liddles test (P = .0002). CONCLUSIONS ARMC5 mutations are implicated in clinically severe Cushings syndrome associated with MAH. Knowledge of a patients ARMC5 status has important clinical implications for the diagnosis of Cushings syndrome and genetic counseling of patients and their families.

X-linked acrogigantism syndrome: clinical profile and therapeutic responses.

X-linked acrogigantism (X-LAG) is a new syndrome of pituitary gigantism, caused by microduplications on chromosome Xq26.3, encompassing the gene GPR101, which is highly upregulated in pituitary tumors. We conducted this study to explore the clinical, radiological, and hormonal phenotype and responses to therapy in patients with X-LAG syndrome. The study included 18 patients (13 sporadic) with X-LAG and microduplication of chromosome Xq26.3. All sporadic cases had unique duplications and the inheritance pattern in two families was dominant, with all Xq26.3 duplication carriers being affected. Patients began to grow rapidly as early as 2-3 months of age (median 12 months). At diagnosis (median delay 27 months), patients had a median height and weight standard deviation scores (SDS) of >+3.9 SDS. Apart from the increased overall body size, the children had acromegalic symptoms including acral enlargement and facial coarsening. More than a third of cases had increased appetite. Patients had marked hypersecretion of GH/IGF1 and usually prolactin, due to a pituitary macroadenoma or hyperplasia. Primary neurosurgical control was achieved with extensive anterior pituitary resection, but postoperative hypopituitarism was frequent. Control with somatostatin analogs was not readily achieved despite moderate to high levels of expression of somatostatin receptor subtype-2 in tumor tissue. Postoperative use of adjuvant pegvisomant resulted in control of IGF1 in all five cases where it was employed. X-LAG is a new infant-onset gigantism syndrome that has a severe clinical phenotype leading to challenging disease management.

Pituitary Adenoma With Paraganglioma/Pheochromocytoma (3PAs) and Succinate Dehydrogenase Defects in Humans and Mice

CONTEXT Germline mutations in genes coding succinate dehydrogenase (SDH) subunits A, B, C, and D have been identified in familial paragangliomas (PGLs)/pheochromocytomas (PHEOs) and other tumors. We described a GH-secreting pituitary adenoma (PA) caused by SDHD mutation in a patient with familial PGLs. Additional patients with PAs and SDHx defects have since been reported. DESIGN We studied 168 patients with unselected sporadic PA and with the association of PAs, PGLs, and/or pheochromocytomas, a condition we named the 3P association (3PAs) for SDHx germline mutations. We also studied the pituitary gland and hormonal profile of Sdhb(+/-) mice and their wild-type littermates at different ages. RESULTS No SDHx mutations were detected among sporadic PA, whereas three of four familial cases were positive for a mutation (75%). Most of the SDHx-deficient PAs were either prolactinomas or somatotropinomas. Pituitaries of Sdhb(+/-) mice older than 12 months had an increased number mainly of prolactin-secreting cells and several ultrastructural abnormalities such as intranuclear inclusions, altered chromatin nuclear pattern, and abnormal mitochondria. Igf-1 levels of mutant mice tended to be higher across age groups, whereas Prl and Gh levels varied according to age and sex. CONCLUSION The present study confirms the existence of a new association that we termed 3PAs. It is due mostly to germline SDHx defects, although sporadic cases of 3PAs without SDHx defects also exist. Using Sdhb(+/-) mice, we provide evidence that pituitary hyperplasia in SDHx-deficient cells may be the initial abnormality in the cascade of events leading to PA formation.