Immunohistochemistry-guided sequencing finds mutations in most cortisol-producing adenomas
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The use of an immunohistochemistry‒guided targeted amplicon sequencing approach using formalin-fixed, paraffin-embedded tissue detected somatic mutations in more than 70% of cortisol-producing adenomas, according to study data.
In findings published in The Journal of Clinical Endocrinology & Metabolism, immunohistochemistry (IHC)‒guided targeted amplicon sequencing was able to detect somatic mutations in 72% of cortisol-producing adenomas, an improvement over the conventional snap-frozen tumor tissue approach, which detected somatic mutations in about half of cortisol-producing tumors.
“We had previously applied a first-in-field sequencing approach by utilizing formalin-fixed, paraffin-embedded tissue for immunohistochemistry-guided DNA capture of aldosterone-producing adenomas,” Juilee Rege, PhD, research investigator in molecular & integrative physiology, and Adina Turcu, MD, MS, associate professor of internal medicine at the University of Michigan, told Healio. “This was followed by targeted next-generation sequencing on genes frequently mutated in familial and sporadic primary aldosteronism. This strategy dramatically improved the detection rate of known and novel aldosterone-driver somatic mutations.”
Rege, Turcu and colleagues analyzed formalin-fixed, paraffin-embedded tissue from 77 patients diagnosed with adrenocorticotropin-independent adrenal Cushing syndrome who underwent a unilateral adrenalectomy at the University of Michigan or Vanderbilt University (84.4% women; median age, 52.9 years). The cohort included 32 patients with overt Cushing’s syndrome and 45 with mild autonomous cortisol excess. Researchers isolated the genomic DNA from formalin-fixed, paraffin-embedded cortisol-producing adenomas to identify somatic mutations. A 17-alpha-hydroxylase/17,20-lyase (CYP17A1) and 3-beta-hydroxysteroid dehydrogenase (HSD3B2) IHC‒guided targeted amplicon sequencing approach was performed on each participant’s resected adrenal gland. Both of these enzymes are required for cortisol synthesis.
Of the somatic mutations identified, the most frequently altered genes were CTNNB1, altered in 42.3% of adenomas; PRKACA, altered in 20.5% of adenomas; and GNAS, altered in 9% of adenomas. The most common mutated gene in those with overt Cushing syndrome was PRKACA, whereas CTNNB1 was the most common mutation found in those with mild autonomous cortisol excess.
“The distinct mutation profiles between overt cortisol-producing adenomas and mild autonomous cortisol excess cortisol-producing adenomas indicate that the underlying genetic causes of cortisol excess in these Cushing syndrome subtypes are different,” Rege and Turcu said. “This unique spectrum of mutations in overt Cushing syndrome and mild autonomous cortisol excess would also indicate a distinct steroidogenic enzyme transcriptomic profile, and distinct steroid fingerprints in the peripheral circulation. Such developments could simplify the diagnosis of suspected Cushing syndrome, which is currently a cumbersome, multistep process.”
The group found two unique adenomas in patients with mild autonomous cortisol excess that exhibited distinct regional heterogeneity in HSD3B2 expression. One somatic mutation that had not been previously reported, PRKACA p.P244_K250delinsQ, was observed in sequencing.
Of the cohort, two men diagnosed with mild autonomous cortisol excess had two adjacent tumors identified within the same adrenal gland. Of these men, one exhibited two cortisol-producing adenomas and had positive staining for CYP17A1 in both tumors. The other man’s adrenal had demonstrated one cortisol-producing adenoma and one aldosterone-producing adenoma, with the cortisol-producing adenoma featuring strong positive staining for CYP17A1 and the aldosterone producing adenoma featuring minimal CYP17A1 staining and strong CYP11B2 expression.
Rege and Turcu said future research will be focused on identifying somatic mutations involved in the pathogenesis of adrenal Cushing syndrome that are currently unknown and uncovering their underlying mechanisms causing cortisol excess.
“We anticipate that novel cortisol-driver somatic mutations are likely present in the remaining 28% of cases, and we are currently using whole-exome sequencing to identify pathogenic variants of genes involved in excess cortisol production,” Rege and Turcu said. “Functional characterization is required to identify the genetic basis of the known and novel mutations associated with cortisol-producing adenomas. No mechanistic studies have been performed to confirm the dysregulated cortisol production in cortisol-producing adenomas harboring any mutations other than PRKACA. Lastly, better tools are needed to correctly diagnose and subtype adrenocorticotropin-independent Cushing syndrome.”
For more information:
Juilee Rege, PhD, can be reached at juilee@med.umich.edu.
Adina Turcu, MD, MS, can be reached at aturcu@med.umich.edu.
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