Sotirios Stergiopoulos

Clinical and molecular genetics of patients with the Carney-Stratakis syndrome and germline mutations of the genes coding for the succinate dehydrogenase subunits SDHB, SDHC, and SDHD.

Gastrointestinal stromal tumors (GISTs) may be caused by germline mutations of the KIT and platelet-derived growth factor receptor-α (PDGFRA) genes and treated by Imatinib mesylate (STI571) or other protein tyrosine kinase inhibitors. However, not all GISTs harbor these genetic defects and several do not respond to STI571 suggesting that other molecular mechanisms may be implicated in GIST pathogenesis. In a subset of patients with GISTs, the lesions are associated with paragangliomas; the condition is familial and transmitted as an autosomal-dominant trait. We investigated 11 patients with the dyad of ‘paraganglioma and gastric stromal sarcoma’; in eight (from seven unrelated families), the GISTs were caused by germline mutations of the genes encoding subunits B, C, or D (the SDHB, SDHC and SDHD genes, respectively). In this report, we present the molecular effects of these mutations on these genes and the clinical information on the patients. We conclude that succinate dehydrogenase deficiency may be the cause of a subgroup of GISTs and this offers a therapeutic target for GISTs that may not respond to STI571 and its analogs.

A transgenic mouse bearing an antisense construct of regulatory subunit type 1A of protein kinase A develops endocrine and other tumours: comparison with Carney complex and other PRKAR1A induced lesions

Background: Inactivation of the human type Iα regulatory subunit (RIα) of cyclic AMP dependent protein kinase (PKA) (PRKAR1A) leads to altered kinase activity, primary pigmented nodular adrenocortical disease (PPNAD), and sporadic adrenal and other tumours. Methods and results: A transgenic mouse carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline responsive promoter (the Tg(Prkar1a*x2as)1Stra, Tg(tTAhCMV)3Uh or tTA/X2AS line) developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia and other features reminiscent of PPNAD, including late onset weight gain, visceral adiposity, and non-dexamethasone suppressible hypercorticosteronaemia, with histiocytic, epithelial hyperplasias, lymphomas, and other mesenchymal tumours. These lesions were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIβ protein levels; the latter biochemical and protein changes were also documented in Carney complex tumours associated with PRKAR1A inactivating mutations and chromosome 17 PRKAR1A locus changes. Conclusion: We conclude that the tTA/X2AS mouse line with a downregulated Prkar1a gene replicates several of the findings in Carney complex patients and their affected tissues, supporting the role of RIα as a candidate tumour suppressor gene.

Down-Regulation of Regulatory Subunit Type 1A of Protein Kinase A Leads to Endocrine and Other Tumors

Mutations of the human type Iα regulatory subunit (RIα) of cyclic AMP-dependent protein kinase (PKA; PRKAR1A) lead to altered kinase activity, primary pigmented nodular adrenocortical disease, and tumors of the thyroid and other tissues. To bypass the early embryonic lethality of Prkar1a−/− mice, we established transgenic mice carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline-responsive promoter. Down-regulation of Prkar1a by up to 70% was achieved in transgenic mouse tissues and embryonic fibroblasts, with concomitant changes in kinase activity and increased cell proliferation, respectively. Mice developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia, and other features reminiscent of primary pigmented nodular adrenocortical disease, histiocytic and epithelial hyperplasias, lymphomas, and other mesenchymal tumors. These were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIβ protein levels. This mouse provides a novel, useful tool for the investigation of cyclic AMP, RIα, and PKA functions and confirms the critical role of Prkar1a in tumorigenesis in endocrine and other tissues.

Human tumors associated with Carney complex and germline PRKAR1A mutations: a protein kinase A disease!

Carney complex (CNC) is a multiple neoplasia syndrome that consists of endocrine (thyroid, pituitary, adrenocortical and gonadal), non‐endocrine (myxomas, nevi and other cutaneous pigmented lesions), and neural (schwannomas) tumors. Primary pigmented nodular adrenocortical disease (PPNAD) is the most common endocrine manifestation of CNC and the only inherited form of Cushing syndrome known to date. In the search of genes responsible for CNC, two chromosomal loci were identified; one (17q22–24) harbored the gene encoding the type I‐α regulatory subunit (RIα) of protein kinase A (PKA), PRKAR1A, a critical component of the cAMP signaling pathway. Here we review CNC and the implications of this discovery for the cAMP and/or PKAs involvement in human tumorigenesis.

Loss of expression of protein kinase A regulatory subunit 1α in pigmented epithelioid melanocytoma but not in melanoma or other melanocytic lesions

Pigmented epithelioid melanocytoma (PEM) is a recently described entity comprising most cases previously described as “animal-type melanoma” and epithelioid blue nevus (EBN) occurring in patients with the multiple neoplasia syndrome Carney complex (CNC). Mutations of the protein kinase A regulatory subunit type 1α (R1α) (coded by the PRKAR1A gene) are found in more than half of CNC patients. In this study, we investigated whether PEM and EBN are related at the molecular level, and whether changes in the PRKAR1A gene status and the expression of the R1α protein may be involved in the pathogenesis of PEM and other melanocytic lesions. Histologic analysis of hematoxylin and eosin-stained sections and immunohistochemistry (IHC) with R1α antibody were performed on 34 sporadic PEMs, 8 CNC-associated PEMs from patients with known PRKAR1A mutations, 297 benign and malignant melanocytic tumors (127 conventional sections of 10 compound nevi, 10 Spitz nevi, 5 deep-penetrating nevi, 5 blue nevi, 6 cellular blue nevi, 2 malignant blue nevi, 3 lentigo maligna, and 86 melanomas of various types); in addition, 170 tissue microarray sections consisting of 35 benign nevi, 60 primary melanomas, and 75 metastatic melanomas, and 5 equine dermal melanomas, were examined. Histologic diagnoses were based on preexisting pathologic reports and were confirmed for this study. DNA studies [loss of heterozygosity (LOH) for the 17q22-24 locus and the PRKAR1A gene sequencing] were performed on 60 melanomas and 7 PEMs. IHC showed that R1α was expressed in all but one core from tissue microarrays (169/170), and in all 127 melanocytic lesions evaluated in conventional sections. By contrast, R1α was not expressed in the 8 EBN from patients with CNC and PRKAR1A mutations. Expression of R1α was lost in 28 of 34 PEMs (82%). R1α was expressed in the 5 equine melanomas studied. DNA studies correlated with IHC findings: there were no PRKAR1A mutations in any of the melanomas studied and the rate of LOH for 17q22-24 was less than 7%; 5 of the 7 PEMs showed extensive 17q22-24 LOH but no PRKAR1A mutations. The results support the concept that PEM is a distinct melanocytic tumor occurring in a sporadic setting and in the context of CNC. They also suggest that PEM differs from melanomas in equine melanotic disease, further arguing that the term animal-type melanoma may be a misnomer for this group of lesions. Loss of expression of R1α offers a useful diagnostic test that helps to distinguish PEM from lesions that mimic it histologically.

Pituitary pathology in Carney complex patients.

Carney complex (CNC) is a familial multiple neoplasia syndrome with features overlapping those of McCune-Albright syndrome (MAS) and multiple endocrine neoplasia (MEN) type 1 (MEN 1). Like MAS and MEN 1 patients, patients with CNC develop growth hormone (GH)-producing pituitary tumors. Occasionally, these tumors are also prolactin-producing, but there are no isolated prolactinomas or other types of pituitary tumors. In at least some patients with CNC, the pituitary gland is characterized by hyperplastic areas; hyperplasia appears to involve somatomammotrophs only. Hyperplasia most likely precedes the formation of GH-producing adenomas in CNC, as has been suggested in MAS-related somatotropinomas, but has never been seen in MEN 1 patients. In at least one case of a patient with CNC and advanced acromegaly, a GH-producing macroadenoma showed extensive genetic changes at the chromosomal level. So far, half of the patients with CNC have germline inactivating mutations in the PRKAR1A gene; in their pituitary tumors, the normal allele of the PRKAR1A gene is lost. Loss-of-hererozygosity suggests that PRKAR1A, which codes for the regulatory subunit type 1α of the cAMP-dependent protein kinase A (PKA) may act as a tumor-suppressor gene in CNC somatomammotrophs. These data provide evidence for a PRKAR1A-induced somatomammotroph hyperpasia in the pituitary tissue of CNC patients; hyperplasia, in turn may lead to additional genetic changes at the somatic level, which then cause the formation of adenomas in some, but not all, patients.

A pleiomorphic GH pituitary adenoma from a Carney complex patient displays universal allelic loss at the protein kinase A regulatory subunit 1A (PRKARIA) locus

Carney complex (CNC) is a familial multiple endocrine neoplasia syndrome associated with GH-producing pituitary tumours and transmitted as an autosomal dominant trait. Mutations of the PRKAR1A gene are responsible for approximately half the known CNC cases but have never found in sporadic pituitary tumours. Pituitary tissue was obtained from an acromegalic CNC patient heterozygote for a common (PRKARIA)i-inactivating mutation. Both immunohistochemistry and electron microscopy showed a highly pleiomorphic pituitary adenoma. The cell culture population appeared morphologically heterogeneous and remained so after more than 30 passages. The mixture was comprised of cells strongly immunostained for GH, spindle-shaped myofibroblast-like cells, and cuboid cells with large axonal projections (negative for GH). The population appeared to have both epithelial and mesenchymal cells. Both at baseline and at passage 30, cytogenetic analysis indicated the presence of normal 46, XY diploid karyotype, whereas losses of the PRKARIAi locus were demonstrated in more than 98% of the cells by fluorescent in situ hybridisation, supporting this gene’s involvement in pituitary tumorigenesis. Allelic loss may have occurred in a single precursor cell type that differentiated and clonally expanded into several phenotypes. Epithelial-to-mesenchymal transition may also occur in CNC-associated pleiomorphic pituitary adenomas.

Functioning paraganglioma and gastrointestinal stromal tumor of the jejunum in three women: Syndrome or coincidence

Functioning paraganglioma and gastrointestinal stromal tumor (GIST) are uncommon tumors that occur mostly in a sporadic and isolated form, occasionally as components of multiple neoplasia syndromes, either separately or together. Separately, they occur in several inherited syndromes including multiple endocrine neoplasia 2, and the GIST, lentigines, and mast cell tumor syndrome. Together, they are variably prominent components of three syndromes: the familial paraganglioma and gastric GIST syndrome, neurofibromatosis type 1, and the Carney triad. The two former conditions are inherited as autosomal dominant traits; the latter does not appear to be inherited and affects young women predominantly. This article reports the nonfamilial occurrence of functioning paraganglioma and GIST of the jejunum in 3 women, 1 young (22 years) at initial presentation. The occurrences were unexpected because of the infrequency of the tumors. The neoplasms, respectively, did not show germline SDHA, SDHB, SDHC, and SDHD, and KIT mutations associated with familial paraganglioma and familial GIST. The paraganglioma-jejunal GIST combination may be the harbinger of a rare genetic syndrome, a variant of the Carney triad or the paraganglioma-gastric stromal sarcoma syndrome, or be coincidental.

Molecular and immunohistochemical investigation of protein kinase a regulatory subunit type 1A (PRKAR1A) in odontogenic myxomas.

Odontogenic myxomas are rare benign neoplasms affecting the jaw. Myxomas of bones and other sites occur as part of Carney complex (CNC), a multiple neoplasia syndrome caused by mutations in the PRKAR1A gene, which codes for the regulatory subunit of protein kinase A (PKA). In the present study, 17 odontogenic myxomas from patients without CNC were screened for PRKAR1A mutations and PRKAR1A protein expression by immunohistochemistry (IHC). Mutations of the coding region of the PRKAR1A gene were identified in 2 tumors; both these lesions showed no or significantly decreased immunostaining of PRKAR1A in the tumor compared to that in the surrounding normal tissue. One mutation (c.725C>A) led to a nonconservative amino acid substitution in a highly conserved area of the gene (A213D); the other was a single base‐pair deletion that led to a frameshift (del774C) and a stop codon 11 amino acids downstream of the mutation site; both tumors were heterozygous for the respective mutations. Of the remaining tumors, 7 of the 15 without mutations showed almost no PRKAR1A in the tumor cells, whereas IHC showed that the protein was abundant in nontumorous cells. We concluded that PRKAR1A may be involved by its down‐regulation in the pathogenesis of odontogenic myxomas caused by mutations and/or other genetic mechanisms. Of the sporadic, nonfamilial tumors associated with PRKAR1A mutations, the odontogenic type was the first myxomatous lesion found to harbor somatic PRKAR1A sequence changes. Published 2005 Wiley‐Liss, Inc.

A mouse model for Carney complex.

Mice with complete inactivation of the type Iα regulatory subunit (RIα) of cyclic (c) AMP‐dependent protein kinase (PKA) (coded by the Prkar1a gene) die early in embryonic life. To bypass the early embryonic lethality of Prkar1a− / − mice, we established transgenic mice carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline‐responsive promoter. Mice developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia, and other features reminiscent of PPNAD, and histiocytic and epithelial hyperplasias, lymphomas, and other mesenchymal tumors. This mouse provides a useful tool for the investigation of cAMP, RIα, and PKA functions and confirms Prkar1as critical role in tumorigenesis in endocrine and other tissues.