Sunitinib in the therapy of malignant paragangliomas: report on the efficacy in a SDHB mutation carrier and review of the literature

SUMMARY Metastatic pheochromocytomas (PHEOs) and paragangliomas (sPGLs) are rare neural crest-derived tumors with a poor prognosis. About 50% of them are due to germ-line mutations of the SDHB gene. At present, there is no cure for these tumors. Their therapy is palliative and represented by different options among which antiangiogenic drugs, like sunitinib, have been hypothesized to be effective especially in malignant SDHB mutated tumors. We report the effects of sunitinib therapy in a SDHB mutation carrier affected by a malignant sPGL. During 101 weeks of therapy at different doses, sunitinib was able to cause a partial response and then a stable disease for a total of 78 weeks. This favorable response is the longest, out of the 35 so far reported in the literature, registered in a patient treated exclusively with sunitinib but, similarly to the other responses, the effect was limited in time. From our analysis of the scanty data present in the literature, the effect of sunitinib does not seem to be different among wild-type patients and those carrying a cluster 1 germ-line mutation. Sunitinib seems able to slow the disease progression in some patients with malignant PHEO/PGL and therefore may represent a therapeutic option, although randomized controlled studies are needed to assess its efficacy definitively in the treatment of these aggressive tumors.

Sunitinib in the therapy of malignant paragangliomas: report on the efficacy in a SDHB mutation carrier and review of the literature.

Metastatic pheochromocytomas (PHEOs) and paragangliomas (sPGLs) are rare neural crest-derived tumors with a poor prognosis. About 50% of them are due to germ-line mutations of the SDHB gene. At present, there is no cure for these tumors. Their therapy is palliative and represented by different options among which antiangiogenic drugs, like sunitinib, have been hypothesized to be effective especially in malignant SDHB mutated tumors. We report the effects of sunitinib therapy in a SDHB mutation carrier affected by a malignant sPGL. During 101 weeks of therapy at different doses, sunitinib was able to cause a partial response and then a stable disease for a total of 78 weeks. This favorable response is the longest, out of the 35 so far reported in the literature, registered in a patient treated exclusively with sunitinib but, similarly to the other responses, the effect was limited in time. From our analysis of the scanty data present in the literature, the effect of sunitinib does not seem to be different among wild-type patients and those carrying a cluster 1 germ-line mutation. Sunitinib seems able to slow the disease progression in some patients with malignant PHEO/PGL and therefore may represent a therapeutic option, although randomized controlled studies are needed to assess its efficacy definitively in the treatment of these aggressive tumors.

Pitfalls in genetic analysis of pheochromocytomas/paragangliomas-case report.

CONTEXT: About 35% of patients with pheochromocytoma/paraganglioma carry a germline mutation in one of the 10 main susceptibility genes. The recent introduction of next-generation sequencing will allow the analysis of all these genes in one run. When positive, the analysis is generally unequivocal due to the association between a germline mutation and a concordant clinical presentation or positive family history. When genetic analysis reveals a novel mutation with no clinical correlates, particularly in the presence of a missense variant, the question arises whether the mutation is pathogenic or a rare polymorphism. OBJECTIVE: We report the case of a 35-year-old patient operated for a pheochromocytoma who turned out to be a carrier of a novel SDHD (succinate dehydrogenase subunit D) missense mutation. With no positive family history or clinical correlates, we decided to perform additional analyses to test the clinical significance of the mutation. METHODS: We performed in silico analysis, tissue loss of heterozygosity analysis, immunohistochemistry, Western blot analysis, SDH enzymatic assay, and measurement of the succinate/fumarate concentration ratio in the tumor tissue by tandem mass spectrometry. RESULTS: Although the in silico analysis gave contradictory results according to the different methods, all the other tests demonstrated that the SDH complex was conserved and normally active. We therefore came to the conclusion that the variant was a nonpathogenic polymorphism. CONCLUSIONS: Advancements in technology facilitate genetic analysis of patients with pheochromocytoma but also offer new challenges to the clinician who, in some cases, needs clinical correlates and/or functional tests to give significance to the results of the genetic assay.

Succinate dehydrogenase subunit B mutations modify human neuroblastoma cell metabolism and proliferation.

Paragangliomas (PGLs) are rare neuroendocrine tumours. About 30-40 % of these tumours are mutated in one of the different susceptibility genes, including those encoding the different subunits of the succinate dehydrogenase, a complex involved both in the tricarboxylic acid cycle and in the oxygen transport chain. The aim of this work was to investigate whether SDHB mutations may account for alterations in cell metabolism and functions. Since human PGL cell lines are not available, we used the neuroblastoma cell line (SK-N-AS) stably transfected with the wild-type human SDHB or different SDHB-mutated constructs carrying some significant mutations found in our patients affected by PGLs. Similarly to succinate dehydrogenase (SDH)-mutated tumour cells, mutated SK-N-AS clones showed reduced SDH enzyme activity. All clones showed normal citrate synthase activity, reduced oxygen consumption and reduced carbonic anhydride production, thus demonstrating a decreased in mitochondrial metabolism. In two of the three mutated SK-N-AS, we also found an increase in HIF1α expression. Surprisingly and unexpectedly, in all the SDHB-mutated clones, we found a significant decrease in glucose uptake and in lactate culture medium concentration, suggesting also a decrease of cytosolic metabolism. Finally, we found that these energetic changes were associated to an increase in cell proliferation and migration. Overall, these data demonstrate that although SDHB mutations significantly downregulate both mitochondrial and cytoplasmic cellular metabolism, these mutations are associated to an upregulation of some cellular functions, such as growth rate and invasiveness.

Patient affected by neurofibromatosis type 1 and thyroid C-cell hyperplasia harboring pathogenic germ-line mutations in both NF1 and RET genes.

Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease with an estimated incidence of 1 in 3000/3500 live births. NF1 is caused by a mutation in a gene which encodes a protein known as neurofibromin. In up to 5% of cases, NF1 is associated with pheochromocytomas. RET proto-oncogene encodes a member of the receptor tyrosine kinase family involved in the normal development or the neoplastic growth of neural crest cell lineages. Germ-line RET mutations account for cases of Multiple Endocrine Neoplasia type 2 (MEN2), an autosomal dominant genetic syndrome where medullary thyroid carcinoma (MTC) is the major and more clinically severe feature, with nearly complete penetrance. C-cell hyperplasia (CCH) is described in MEN2 patients, and it has been implicated as the precursor of in situ MTC. Patients with RET mutations develop pheochromocytomas in 50% of cases. Rarely, patients with NF1 have been found to present, in addition to the NF1 clinical picture, other lesions, such as parathyroid hyperplasia/adenoma and/or medullary thyroid carcinoma. In spite of the presence of these MEN2 lesions, in none of these patients mutations of gene RET have been found so far. In this report, we describe the first case of a patient affected by a germ-line mutation in both NF1 and RET genes.

Updated and new perspectives on diagnosis, prognosis, and therapy of malignant pheochromocytoma/paraganglioma.

Malignant pheochromocytomas/paragangliomas are rare tumors with a poor prognosis. Malignancy is diagnosed by the development of metastases as evidenced by recurrences in sites normally devoid of chromaffin tissue. Histopathological, biochemical, molecular and genetic markers offer only information on potential risk of metastatic spread. Large size, extraadrenal location, dopamine secretion, SDHB mutations, a PASS score higher than 6, a high Ki-67 index are indexes for potential malignancy. Metastases can be present at first diagnosis or occur years after primary surgery. Measurement of plasma and/or urinary metanephrine, normetanephrine and metoxytyramine are recommended for biochemical diagnosis. Anatomical and functional imaging using different radionuclides are necessary for localization of tumor and metastases. Metastatic pheochromocytomas/paragangliomas is incurable. When possible, surgical debulking of primary tumor is recommended as well as surgical or radiosurgical removal of metastases. I-131-MIBG radiotherapy is the treatment of choice although results are limited. Chemotherapy is reserved to more advanced disease stages. Recent genetic studies have highlighted the main pathways involved in pheochromocytomas/paragangliomas pathogenesis thus suggesting the use of targeted therapy which, nevertheless, has still to be validated. Large cooperative studies on tissue specimens and clinical trials in large cohorts of patients are necessary to achieve better therapeutic tools and improve patient prognosis.

Genetic-clinical profile of subjects with apparently sporadic extra-adrenal paragangliomas.

BACKGROUND: Mutations in the genes encoding B, C, and D subunits of the succinate dehydrogenase (SDH) are involved in the pathogenesis of familial paraganglioma (PGL) syndrome. Many subjects with apparently sporadic extra-adrenal paragangliomas are found to be carrier for SDH mutation. OBJECTIVE: Here we describe four subjects with apparently sporadic extra-adrenal paragangliomas with newly identified mutations in the SDH subunit B and the related clinical phenotype. METHODS: Gene sequencing was performed to search for mutations in the SDHB (all exons), SDHC (all exons), and SDHD (all exons) genes as well as VHL (all exons) and RET (10, 11, 13, 14, 15, 16 exons) genes in all four index cases. A complete clinical, biochemical, and instrumental work-up was performed. RESULTS: Three subjects were found to be affected with a nonsense SDHB germline mutation (Q30X, Y61X, and W201X, respectively). These mutations are predicted to encode for a truncated SDHB protein. The fourth subject presented a S195del frameshift mutation, causing a deletion of the codon AGC, encoding for a serine. Clinical presentation and course of each patient is described. CONCLUSIONS: Extra-adrenal paragangliomas, localized in the sympathetic ganglia (in the posterior thorax or in the abdomen), are very often SDHB-inherited form rather than sporadic tumor. Our data confirm the importance of genetic screening in patients affected with paragangliomas and enlarge the list of mutations responsible for the presence of these tumors.

Head and neck paragangliomas: genetic spectrum and clinical variability in 79 consecutive patients.

Head and neck paragangliomas (HNPGLs) are neural crest-derived tumors. In comparison with paragangliomas located in the abdomen and the chest, which are generally catecholamine secreting (sPGLs) and sympathetic in origin, HNPGLs are, in fact, parasympathetic in origin and are generally nonsecreting. Overall, 79 consecutive patients with HNPGL were examined for mutations in SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, MAX, and TMEM127 genes by PCR/sequencing. According to a detailed family history (FH) and clinical, laboratory (including metanephrines), and instrumental examinations, patients were divided into three groups: a) patients with a positive FH for HNPGL (index cases only), b) patients with a negative FH and multiple HNPGLs (synchronous or metachronous) or HNPGL associated with an sPGL, and c) patients with negative FH and single HNPGL. The ten patients in group a) proved to be SDHD mutation carriers. The 16 patients in group b) proved to be SDHD mutation carriers. Among the 53 patients in group c), ten presented with germ-line mutations (three SDHB, three SDHD, two VHL, and two SDHAF2). An sPGL was found at diagnosis or followed up in five patients (6.3%), all were SDHD mutation carriers. No SDHC, SDHA, MAX, and TMEM127 mutations were found. In SDHD mutation carriers, none of the patients affected by HNPGL associated with sPGL presented missense mutations. In conclusion, a positive FH or the presence of multiple HNPGLs is a strong predictor for germ-line mutations, which are also present in 18.8% of patients carefully classified as sporadic. The most frequently mutated gene so far is SDHD but others, including SDHB, SDHAF2, and VHL, may also be affected.

Functional study in a yeast model of a novel succinate dehydrogenase subunit B gene germline missense mutation (C191Y) diagnosed in a patient affected by a glomus tumor.

Mutations of succinate dehydrogenase (SDH) subunits B, C and D are associated to pheochromocytoma/paraganglioma (PGL) development. The mechanisms linking SDH mutations to tumorigenesis are currently unknown. We report a novel germline missense SDHB mutation (C191Y) in a patient affected by a glomus tumor. The missense mutation hits an amino acid residue conserved from mammals to the yeast Saccharomyces cerevisiae. The pathogenic significance of the human mutation was validated in a yeast model. SDH2(C184Y) mutant allele equivalent to human SDHB(C191Y) did not restore the OXPHOS phenotype of the Deltasdh2 null mutant. In the mutant, SDH activity was also abolished along with a reduction in respiration. Sensitivity to oxidative stress was increased in the mutant, as revealed by reduced growth in the presence of menadione. Remarkably, the frequency of petite colony formation was increased in the mutant yeast strain, indicating an increased mtDNA mutability. Histochemistry demonstrates that SDH activity was selectively absent in the patient tumor tissue. Overall, our results demonstrate that the C191Y SDHB mutation suppresses SDH enzyme activity leading to increased ROS formation and mtDNA mutability in our yeast model. These findings further our understanding of the mechanisms underlying PGL development and point to the yeast model as a valid tool to investigate on the possible pathogenic relevance of SDH novel mutations and/or rare polymorphism.

Clinically guided genetic screening in a large cohort of italian patients with pheochromocytomas and/or functional or nonfunctional paragangliomas.

PURPOSE: The aim of the study was to define the frequency of hereditary forms and the genotype/phenotype correlations in a large cohort of Italian patients with pheochromocytomas and/or functional or nonfunctional paragangliomas. DESIGN: We examined 501 consecutive patients with pheochromocytomas and/or paragangliomas (secreting or nonsecreting). Complete medical and family histories, as well as the results of clinical, laboratory, and imaging studies, were recorded in a database. Patients were divided into different groups according to their family history, the presence of lesions outside adrenals/paraganglia considered syndromic for VHL disease, MEN2, and NF1, and the number and types of pheochromocytomas and/or paragangliomas. Germ-line mutations in known susceptibility genes were investigated by gene sequencing (VHL, RET, SDHB, SDHC, SDHD) or diagnosed according to phenotype (NF1). In 160 patients younger than 50 yr with a wild-type profile, multiplex ligation-dependent probe amplification assays were performed to detect genomic rearrangements. RESULTS: Germline mutations were detected in 32.1% of cases, but frequencies varied widely depending on the classification criteria and ranged from 100% in patients with associated syndromic lesions to 11.6% in patients with a single tumor and a negative family history. The types and number of pheochromocytomas/paragangliomas as well as age at presentation and malignancy suggest which gene should be screened first. Genomic rearrangements were found in two of 160 patients (1.2%). CONCLUSIONS: The frequency of the hereditary forms of pheochromocytoma/paraganglioma varies depending on the family history and the clinical presentation. A positive family history and an accurate clinical evaluation of patients are strong indicators of which genes should be screened first.