Corticotropinoma as a Component of Carney Complex

Abstract

Known germline gene abnormalities cause one-fifth of the pituitary adenomas in children and adolescents, but, in contrast with other pituitary tumor types, the genetic causes of corticotropinomas are largely unknown. In this study, we report a case of Cushing disease (CD) due to a loss-of-function mutation in PRKAR1A, providing evidence for association of this gene with a corticotropinoma. A 15-year-old male presenting with hypercortisolemia was diagnosed with CD. Remission was achieved after surgical resection of a corticotropin (ACTH)-producing pituitary microadenoma, but recurrence 3 years later prompted reoperation and radiotherapy. Five years after the original diagnosis, the patient developed ACTH-independent Cushing syndrome, and a diagnosis of primary pigmented nodular adrenocortical disease was confirmed. A PRKAR1A mutation (c.671delG, p.G225Afs*16) was detected in a germline DNA sample from the patient, which displayed loss of heterozygosity in the corticotropinoma. No other germline or somatic mutations of interest were found. As corticotropinomas are not a known component of Carney complex (CNC), we performed loss of heterozygosity and messenger RNA stability studies in the patient's tissues, and analyzed the effect of Prkar1a silencing on AtT-20/D16v-F2 mouse corticotropinoma cells. No PRKAR1A defects were found among 97 other pediatric CD patients studied. Our clinical case and experimental data support a role for PRKAR1A in the pathogenesis of a corticotroph cell tumor. This is a molecularly confirmed report of a corticotropinoma presenting in association with CNC. We conclude that germline PRKAR1A mutations are a novel, albeit apparently infrequent, cause of CD.

Keywords: Carney complex; Cushing disease; genetics; pituitary tumor; protein kinase A.

Figure 1.

Clinical and histopathological presentation. (a and b) Small epicanthal lentigines were observed in this patient. (c) The surgical specimens of bilateral adrenalectomy displayed the characteristics of PPNAD, and such diagnosis was later confirmed by histopathological examination. (d) Hematoxylin–eosin staining (20×) of the corticotropinoma tissue. The tumor was a microadenoma measuring approximately 6 × 4 × 2 mm, with Crooke’s cells surrounding the neoplastic tissue. (e) Breakdown of the reticulin network (20×), (f) as well as strong and diffusely positive ACTH staining (20×), was demonstrated. (g) Extensive positive immunostaining for CAM5.2 was identified (20×). (h) Keratin 20 immunostaining was found in some areas containing Crooke’s cells (20×). These images were compatible with a diagnosis of Crooke’s cell adenoma.

Figure 2.

Role of PRKAR1A in corticotroph cell tumorigenesis, and in PPNAD. (a) The frameshift PRKAR1A gene (NG_007093.3) variant c.671delG, p.G225Afs*16 affects the exon 7 of the reference transcript (NM_002734.4, the first exon is not translated). The surrounding region encodes the first of two cyclic adenosine monophosphate–binding domains in the protein, which are crucial for its regulatory function. (b) The germline mutation identified in the patient was not present in the mother. As a sample from the father was not available, we could not determine whether the mutation was inherited from the father or if it appeared as a de novo event. The PPNAD and nonadenomatous pituitary (obtained from the second surgery) tissues were heterozygous for such mutation. However, loss of heterozygosity was identified in the corticotropinoma tissue, with a 72% to 82% predominance of mutant DNA in the chromatogram peaks measured. (c) In samples from lymphoblastoid cells before and after the treatment with cycloheximide and in the PPNAD tissue, only the wild-type allele was detected. Given that these tissues did not display loss of heterozygosity at the DNA level, the homozygosity for the wild-type allele should be explained by nonsense-mediated messenger RNA decay. Unfortunately, we did not achieve rescue of the mutant allele during the cycloheximide experiment performed. (d) Compared with a normal adrenal tissue sample, the PPNAD specimen displayed significantly reduced Prkar1a expression (mean: 1 ± 0.02 vs 0.68 ± 0.01, P < 0.0001). (e) We achieved 30% Prkar1a KD compared with the scrambled control (mean: 0.68 ± 0.02 vs 1 ± 0.01, P < 0.0001 KD). Compared with the untransfected cells, Trp53 expression was reduced in the KD experiment (mean: 1.05 ± 0.03 vs 0.91 ± 0.1, P = 0.0130), and there was a trend for lower Trp53 in the KD cells compared with the scrambled control (mean: 1 ± 0.05 vs 0.91 ± 0.1, P = 0.13). No other significant differences in the expression of cell cycle markers were found among experimental conditions.