Targeted Mutational Analysis of Cortisol-Producing Adenomas

Abstract

Context: Somatic gene mutations have been identified in only about half of cortisol-producing adenomas (CPAs). Affected genes include PRKACA, GNAS, PRKAR1A, and CTNNB1.

Objective: This work aims to expand our understanding of the prevalence of somatic mutations in CPAs from patients with overt Cushing syndrome (OCS) and "subclinical" mild autonomous cortisol excess (MACE), with an immunohistochemistry (IHC)‒guided targeted amplicon sequencing approach using formalin-fixed paraffin-embedded (FFPE) tissue.

Methods: We analyzed FFPE adrenal tissue from 77 patients (n = 12 men, 65 women) with either OCS (n = 32) or MACE (n = 45). Using IHC for 17α-hydroxylase/17,20-lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase (HSD3B2), we identified 78 CPAs (32 OCS CPAs and 46 MACE CPAs). Genomic DNA was isolated from the FFPE CPAs and subjected to targeted amplicon sequencing for identification of somatic mutations.

Results: Somatic mutations were identified in 71.8% (56/78) of the CPAs. While PRKACA was the most frequently mutated gene in OCS CPAs (14/32, 43.8%), somatic genetic aberrations in CTNNB1 occurred in 56.5% (26/46) of the MACE CPAs. Most GNAS mutations were observed in MACE CPAs (5/7, 71.4%). No mutations were observed in PRKAR1A. In addition to the known mutations, we identified one previously unreported mutation in PRKACA. Two patients with MACE harbored 2 adjacent tumors within the same adrenal gland - one patient had 2 CPAs, and the other patient had a CPA and an aldosterone-producing adenoma (identified by IHC for aldosterone synthase).

Conclusion: A comprehensive FFPE IHC-guided gene-targeted sequencing approach identified somatic mutations in 71.8% of the CPAs. OCS CPAs demonstrated a distinct mutation profile compared to MACE CPAs.

Keywords: Cushing syndrome; cortisol-producing adenoma; mild autonomous cortisol excess; mutational analysis; targeted amplicon sequencing.

Figure 1.

Prevalence of somatic mutations in 78 cortisol-producing adenomas (CPAs) reported in 77 patients with adrenal Cushing syndrome (CS). Of the 77 patients, 32 were diagnosed with overt CS and 45 with mild adrenal cortisol excess (MACE). The candidate genes tested were PRKACA, CTNNB1, GNAS, and PRKAR1A.

Figure 2.

Histopathologic characteristics of cortisol-producing adenomas (CPAs) with CTNNB1 (p.S45P), PRKACA (p.L206R) , and GNAS (p.R201H) somatic mutations. A to C, G to I, and M to O, Scans, and D to F, J to L, and P to R high-magnification images of the tumors are presented. A, D, G, J, M, and P, Hematoxylin and eosin (HE) staining; B, E, H, K, N, Q, 17α-hydroxylase/17,20 lyase (CYP17A1); and C, F, I, L, O, R 3β-hydroxysteroid dehydrogenase (HSD3B2) immunohistochemistry. Scale: 5 mm (scans) and 100 µm (high magnification).

Figure 3.

Histopathologic characteristics of cortisol-producing adenomas (CPAs) with a distinct intratumor heterogeneity in 3β-hydroxysteroid dehydrogenase (HSD3B2). The regions with differential expression of HSD3B2 have been labeled as 1, 2, and 3. A, E, and I, Scans, and B to D, F to H, and J to L high-magnification images of the tumors are presented. A to D, Hematoxylin and eosin (HE) staining; E to H, 17α-hydroxylase/17,20 lyase (CYP17A1); and I to L, 3β-hydroxysteroid dehydrogenase (HSD3B2) immunohistochemistry. Scale: 5 mm (scans) and 100 µm (high magnification). Targeted amplicon sequencing indicated that all 3 domains harbored the CTNNB1 p.S45P mutation.

Figure 4.

DNA sequence and histopathologic characteristics of cortisol-producing adenomas (CPAs) harboring the novel PRKACA p.P244_K250delinsQ somatic mutation. A, Sanger sequence chromatogram of the CPA with the PRKACA p.P244_K250delinsQ mutation and the adjacent normal tissue control. CPA sequence: The top row of nucleotides in the yellow box denotes the nucleotide trace in the matched normal adjacent adrenal tissue deleted in the CPA. The codon for “Q” in the yellow box denotes the inserted sequence. B, Hematoxylin and eosin (HE) staining; C, 17α-hydroxylase/17,20 lyase (CYP17A1); and D, 3β-hydroxysteroid dehydrogenase (HSD3B2) immunohistochemistry. Scale: 5 mm.

Figure 5.

Histopathologic findings of a cortisol-producing adenoma (CPA) (T1) and a CYP11B2-positive aldosterone-producing adenoma (APA) (T2) within the same adrenal gland. A, D, G, and J, Scans, and B, C, E, F, H, I, K, and L high-magnification images of the tumors are presented. A to C, Hematoxylin and eosin (HE) staining; D to F, 17α-hydroxylase/17,20 lyase (CYP17A1), G to I, 3β-hydroxysteroid dehydrogenase (HSD3B2); and J to L, aldosterone synthase (CYP11B2) immunohistochemistry. Scale: 5 mm (scans) and 100 µm (high magnification). Targeted amplicon sequencing indicated that the CPA harbored the CTNNB1 p.P44A mutation and the APA expressed the CACNA1D p.R993T mutation.