CYP11B2 Immunohistochemistry Research Articles

FRI137 Histologic And Genetic Characteristics Of Primary Aldosteronism In Late Adulthood

Abstract Disclosure: A. Blinder: None. K. Nanba: None. D. Sapiro: None. J. Baker: None. C. Liu: None. I. Bancos: None. M. Lyden: None. W. Young: None. H. Wachtel: None. D. Cohen: None. J. Luther: None. T.A. Williams: None. M. Reincke: None. T. Else: None. T.J. Giordano: None. A. Udager: None. W.E. Rainey: None. Background: There is accumulating evidence that changes in adrenal histology and regulation of renin-angiotensin-aldosterone system occur with age. Primary aldosteronism (PA) is the most common cause of endocrine hypertension. Recent clinical studies suggest unique characteristics of PA in elderly patients, including high prevalence of comorbidities and low clinical cure rate after adrenalectomy. However, histologic and genetic features of PA in late adulthood have not been well elucidated. Objective: To determine histologic and genetic characteristics of PA in late adulthood. Methods: Seventy-six patients who underwent unilateral adrenalectomy at the age of 60 or older were included in this study. Formalin-fixed, paraffin-embedded (FFPE) sections of resected adrenal tissue were used for the analysis. Aldosterone synthase (CYP11B2) immunohistochemistry (IHC) was performed to identify the source of inappropriate aldosterone production. Histologic classification was made according to the HISTALDO (histopathology of primary aldosteronism) consensus. CYP11B2 IHC-guided DNA capture followed by sequencing analysis using direct Sanger sequencing or ion torrent based next-generation sequencing (NGS) was performed. Results: Median age of the studied patients was 70 (interquartile range: 64-73). Based on the CYP11B2 IHC, histologic diagnosis was made as follows; aldosterone-producing adenoma (APA, n=23), aldosterone-producing nodule (APN, n=41), and non-functioning adrenocortical adenoma (NFA, n=6). The remaining six cases had multiple CYP11B2-expressing neoplastic lesions, including APA with APN (n=2) and multiple APNs (n=4). Co-existence of NFA with APA or APN(s) was also observed in 13. Sequencing analysis revealed that somatic KCNJ5 mutation was the most frequent genetic alteration in APA (7/23, 30%), whereas somatic CACNA1D mutation was the most common genetic cause of APN (20/39 with successful sequencing, 51%). Of the 18 sequenced NFA DNA samples, two had somatic mutations in CTNNB1 (n=1) and GNAS (n=1). Conclusion: APN is the major histologic feature of PA in late adulthood. Co-existence of NFA also appears to be a frequent observation in this age population. Somatic CACNA1D and KCNJ5 mutations are common in APN and APA, respectively. Presentation: Friday, June 16, 2023

Primary Aldosteronism: Spatial Multiomics Mapping of Genotype-Dependent Heterogeneity and Tumor Expansion of Aldosterone-Producing Adenomas.

Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis. Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry. We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (CYP11B2-type 1 or 2). The CYP11B2-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-KCNJ5MUT and APA-KCNJ5WT. The CYP11B2-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-KCNJ5MUT. Metabolites that promote oxidative stress and cell death accumulated in APA-KCNJ5WT. In contrast, antioxidant metabolites were abundant in APA-KCNJ5MUT. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states. Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.

PS-BPB06-9: PREVALENCE OF KCNJ5 MUTANT ALDOSTERONE-PRODUCING ADENOMAS AMONG MALAYSIAN PATIENTS WITH PRIMARY ALDOSTERONISM

Objective: To verify the prevalence of KCNJ5 mutant aldosterone-producing adenomas (APAs) among primary aldosteronism (PA) patients from a tertiary center in Malaysia Design and method: A total of 80 adrenal samples from adrenalectomies performed between 2010 up to 2020 at Hospital Putrajaya, Malaysia were included in this study. Of the adrenals blocks received, DNA samples from 74 APAs identified by CYP11B2 immunohistochemistry (IHC) staining were sent for KCNJ5 mutation analysis. Gender, ethnicity and age at adrenalectomy were compared between genotypes. Data were statistical analyzed using the Fisher Exact test. Results: 28 of the 74 (37.8%) APAs had a KCNJ5 mutation. The most common KCNJ5 variant was G151R (50%), followed by L168R (39.3%) and T158A (10.7%). Of all the mutant APAs, 19/28 (67.9%) were from female PA patients whereas among controls 26/46 (56.5%) were females. No significant association between KCNJ5 mutation status and gender were found. The majority of mutant APAs were from Malay PA patients (n = 17), followed by Chinese PA patients (n = 11) which follows the whole cohort demography. Thus, ethnicity was not found to be significantly associated with KCNJ5 mutation status. In this cohort, no significant differences in age at of adrenalectomy by KCNJ5 mutation status was seen. Adrenalectomy procedure was performed most frequent between the age of 37–46 years old (n = 30), followed by 47–56 (n = 16), 26–35 (n = 12), 57–66 (n = 11) and 67–76 (n = 2); in 3 patients this information was not known. Conclusions: A similar prevalence of KCNJ5 mutant APAs as found in a previous study performed at the National University of Malaysia Medical Centre between 2000 and 2015 verifies the lower prevalence of KCNJ5 mutant APAs in Malaysian PA patients (37.8%) compared to that observed in other Oriental cohorts (up to 70%). Although, the majority of mutant APAs were from female PA patients (67.9%), no gender bias according to KCNJ5 mutation status was found as the majority of PA patients in this cohort were females (60.8%).

PS-BPB06-10: DISTRIBUTION OF CACNA1D SOMATIC MUTATIONS AMONG CYP11B2 IMMUNOHISTOCHEMISTRY-GUIDED SAMPLES OF ALDOSTERONE-PRODUCING ADENOMA (APA)

Objectives: Past studies in Afro-Caribbean primary aldosteronism (PA) patients have found CACNA1D mutations to be the most common aldosterone-driving mutation among their cohort; a striking difference to Caucasian and Oriental cohorts where KCNJ5 mutations are reported to be the most common. However not many studies had used aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided sequencing approach of the whole CACNA1D gene, performing instead unguided adenoma sampling and hotspot sequencing. We thus performed CYP11B2 IHC-guided whole gene sequencing on APAs from an Oriental cohort (Malaysia) and a Caucasian cohort (Czech Republic) for comparison. Design and method: Surgically removed adrenals from 100 Malaysian and Czech PA patients with unilateral disease were studied. Genomic DNA was isolated from adenoma FFPE sections that had a positive CYP11B2 IHC staining and somatic mutations analysis were performed by next generation sequencing (NGS) of known aldosterone-driver genes. Predicted pathogenic mutations were verified by Sanger Sequencing. Results: Out of 100 FFPE samples sent for NGS, 36 were excluded due to the quality of the data. In total 58 of 64 APAs (87.5%) had a pathogenic aldosterone-driver somatic mutation. KCNJ5 mutations were the most common (n = 37, 57.8%), followed by CACNA1D (n = 11, 20.3%), ATP1A1 (n = 6, 9.4%), ATP2B3 (n = 3, 4.7%) and CTNNB1 (n = 2, 3.1%) mutations. Of particular interest, one APA sample had two pathogenic somatic mutations, CACNA1D p.Val1373Met and CTNNB1: p.Thr41Ala. SIFT and Polyphen2 scores predicted both mutations to be deleterious. Both mutations have previously been reported separately to cause APAs. The CACNA1D/CTNNB1 mutant APA, was solitary, with homogeneous CYP11B2 expression while negative for CYP17A1 expression. The APA was adrenalectomized from a 53-year-old male Czech patient. The majority of patients with APAs harboring CACNA1D mutations were male (n = 8, 72.7%) and from the Czech cohort (n = 7, 64%). There were no significant differences in age at adrenalectomy between patients harboring a CACNA1D mutant APA (45 ± 3) and those with a non-CACNA1D mutant APA (48 ± 2). Conclusions: Solitary ZG APAs can harbor both a CACNA1D and a CTNNB1 mutation. The prevalence of CACNA1D in this CYP11B2-IHC guided study is consistent with previous results reported in Oriental and Caucasian cohorts. Although CACNA1D is not the top genetic alteration responsible for APAs in these cohorts, CACNA1D may still be a potential key player in idiopathic hyperaldosteronism and aldosterone-producing micronodules. Thus further study is required to characterize the distribution of CACNA1D pathogenic mutations in bilateral PA.

Functional nodules in primary aldosteronism: identification of CXCR4 expression with 68Ga-pentixafor PET/CT.

We analyzed the diagnostic efficiency of 68Ga-pentixafor PET/CT for functional nodules in primary aldosteronism (PA). Furthermore, we compared the correlation of CXCR4 expression with aldosterone synthase (CYP11B2) expression and PET/CT uptake in these patients. We prospectively assessed 50 patients diagnosed with PA and 10 patients with non-functional adrenal adenoma (NFA). All patients underwent 68Ga-pentixafor PET/CT before adrenalectomy. Immunohistochemistry (IHC) was performed to detect the protein expression of CYP11B2 and the G-protein-coupled receptor CXCR4. CYP11B2 IHC revealed the presence of 43 functional nodules. Subsequently, 40/43 functional nodules could be detected on 68Ga-pentixafor PET/CT, while negative imaging findings were noted for 11/13 non-functional nodules (sensitivity, 93.0%; specificity, 84.6%). The optimum SUVmax cut-off for the identification of functional nodules was 8.95 (AUC 0.914 [0.828-1.000], p < 0.001). Regarding the size of functional nodules, diagnostic efficiency appeared to be much higher for nodules greater than 1 cm in size (sensitivity, up to 97.3%). Moreover, we examined the relationship between CXCR4 and CYP11B2 expression in 56 lesions. All 43 CYP11B2-positive nodules were CXCR4-positive, but one of the 13 CYP11B2-negative nodules (7.7%) showed false-positive staining for CXCR4. Moreover, the consistency between PET/CT uptake and CXCR4 staining results was 92.9% (52/56). At least 90% of functional nodules show positive uptake on 68Ga-pentixafor PET/CT, and the detection ability is much better for nodules with a diameter ≥ 1 cm. With its high sensitivity and specificity, 68Ga-pentixafor PET/CT can be considered a promising surgical decision-making tool for patients with PA. • 68Ga-pentixafor PET/CT could be a useful tool for the identification of functional adrenal nodules in APAs and even IHAs. • The diagnostic efficiency appears to be much higher for nodules ≥ 1 cm in size. • There is high consistency between the results of 68Ga-pentixafor PET/CT imaging and CXCR4 immunohistochemistry.

Histopathology and Genetic Causes of Primary Aldosteronism in Young Adults.

Due to its rare incidence, molecular features of primary aldosteronism (PA) in young adults are largely unknown. Recently developed targeted mutational analysis identified aldosterone-driver somatic mutations in aldosterone-producing lesions, including aldosterone-producing adenomas (APAs), aldosterone-producing nodules (APNs), and aldosterone-producing micronodules, formerly known as aldosterone-producing cell clusters. To investigate histologic and genetic characteristics of lateralized PA in young adults. Formalin-fixed, paraffin-embedded adrenal tissue sections from 74 young patients with lateralized PA (<35 years old) were used for this study. Immunohistochemistry (IHC) for aldosterone synthase (CYP11B2) was performed to define the histopathologic diagnosis. Somatic mutations in aldosterone-producing lesions were further determined by CYP11B2 IHC-guided DNA sequencing. Based on the CYP11B2 IHC results, histopathologic classification was made as follows: 48 APAs, 20 APNs, 2 multiple aldosterone-producing nodules (MAPN), 1 double APN, 1 APA with MAPN, and 2 nonfunctioning adenomas (NFAs). Of 45 APAs with successful sequencing, 43 (96%) had somatic mutations, with KCNJ5 mutations being the most common genetic cause of young-onset APA (35/45, 78%). Of 18 APNs with successful sequencing, all of them harbored somatic mutations, with CACNA1D mutations being the most frequent genetic alteration in young-onset APN (8/18, 44%). Multiple CYP11B2-expressing lesions in patients with MAPN showed several aldosterone-driver mutations. No somatic mutations were identified in NFAs. APA is the most common histologic feature of lateralized PA in young adults. Somatic KCNJ5 mutations are common in APAs, whereas CACNA1D mutations are often seen in APNs in this young PA population.

Overview of the 2022 WHO Classification of Adrenal Cortical Tumors

The new WHO classification of adrenal cortical proliferations reflects translational advances in the fields of endocrine pathology, oncology and molecular biology. By adopting a question–answer framework, this review highlights advances in knowledge of histological features, ancillary studies, and associated genetic findings that increase the understanding of the adrenal cortex pathologies that are now reflected in the 2022 WHO classification. The pathological correlates of adrenal cortical proliferations include diffuse adrenal cortical hyperplasia, adrenal cortical nodular disease, adrenal cortical adenomas and adrenal cortical carcinomas. Understanding germline susceptibility and the clonal-neoplastic nature of individual adrenal cortical nodules in primary bilateral macronodular adrenal cortical disease, and recognition of the clonal-neoplastic nature of incidentally discovered non-functional subcentimeter benign adrenal cortical nodules has led to redefining the spectrum of adrenal cortical nodular disease. As a consequence, the most significant nomenclature change in the field of adrenal cortical pathology involves the refined classification of adrenal cortical nodular disease which now includes (a) sporadic nodular adrenocortical disease, (b) bilateral micronodular adrenal cortical disease, and (c) bilateral macronodular adrenal cortical disease (formerly known primary bilateral macronodular adrenal cortical hyperplasia). This group of clinicopathological entities are reflected in functional adrenal cortical pathologies. Aldosterone producing cortical lesions can be unifocal or multifocal, and may be bilateral with no imaging-detected nodule(s). Furthermore, not all grossly or radiologically identified adrenal cortical lesions may be the source of aldosterone excess. For this reason, the new WHO classification endorses the nomenclature of the HISTALDO classification which uses CYP11B2 immunohistochemistry to identify functional sites of aldosterone production to help predict the risk of bilateral disease in primary aldosteronism. Adrenal cortical carcinomas are subtyped based on their morphological features to include conventional, oncocytic, myxoid, and sarcomatoid subtypes. Although the classic histopathologic criteria for diagnosing adrenal cortical carcinomas have not changed, the 2022 WHO classification underscores the diagnostic and prognostic impact of angioinvasion (vascular invasion) in these tumors. Microscopic angioinvasion is defined as tumor cells invading through a vessel wall and forming a thrombus/fibrin-tumor complex or intravascular tumor cells admixed with platelet thrombus/fibrin. In addition to well-established Weiss and modified Weiss scoring systems, the new WHO classification also expands on the use of other multiparameter diagnostic algorithms (reticulin algorithm, Lin–Weiss–Bisceglia system, and Helsinki scoring system) to assist the workup of adrenal cortical neoplasms in adults. Accordingly, conventional carcinomas can be assessed using all multiparameter diagnostic schemes, whereas oncocytic neoplasms can be assessed using the Lin–Weiss–Bisceglia system, reticulin algorithm and Helsinki scoring system. Pediatric adrenal cortical neoplasms are assessed using the Wieneke system. Most adult adrenal cortical carcinomas show > 5 mitoses per 10 mm2 and > 5% Ki67. The 2022 WHO classification places an emphasis on an accurate assessment of tumor proliferation rate using both the mitotic count (mitoses per 10 mm2) and Ki67 labeling index which play an essential role in the dynamic risk stratification of affected patients. Low grade carcinomas have mitotic rate of ≤ 20 mitoses per 10 mm2, whereas high-grade carcinomas show > 20 mitoses per 10 mm2. Ki67-based tumor grading has not been endorsed in the new WHO classification, since the proliferation indices are continuous variables rather than being static thresholds in tumor biology. This new WHO classification emphasizes the role of diagnostic and predictive biomarkers in the workup of adrenal cortical neoplasms. Confirmation of the adrenal cortical origin of a tumor remains a critical requirement when dealing with non-functional lesions in the adrenal gland which may be mistaken for a primary adrenal cortical neoplasm. While SF1 is the most reliable biomarker in the confirmation of adrenal cortical origin, paranuclear IGF2 expression is a useful biomarker in the distinction of malignancy in adrenal cortical neoplasms. In addition to adrenal myelolipoma, the new classification of adrenal cortical tumors has introduced new sections including adrenal ectopia, based on the potential role of such ectopic tissue as a possible source of neoplastic proliferations as well as a potential mimicker of metastatic disease. Adrenal cysts are also discussed in the new classification as they may simulate primary cystic adrenal neoplasms or even adrenal cortical carcinomas in the setting of an adrenal pseudocyst.

Characteristics of a Novel ATP2B3 K416_F418delinsN Mutation in a Classical Aldosterone-Producing Adenoma.

Simple SummaryThe ATP2B3 channel mutation is a rare cause of primary aldosteronism (PA). ATP2B3 gene mutation leads to the dysfunction of calcium channel that pumps calcium ion out of the cell and accumulates intracellular calcium signal to stimulate aldosterone synthesis. In the present study, we found a novel somatic ATP2B3 K416_F418delinsN mutation in a PA patient, and proved its functionality by demonstrating aldosterone hyper-function in the mutant-transfected adrenal cell-line. The ATP2B3 K416_F418delinsN mutation resulted from the deletion from nucleotides 1248 to 1253. The translated amino acid sequence from 416 to 418 as lysine-phenylalanine-phenylalanine was deleted and an asparagine was inserted due to the merging of residual nucleotide sequences.In patients with primary aldosteronism (PA), the prevalence of ATP2B3 mutation is rare. The aim of this study is to report a novel ATP2B3 mutation in a PA patient. Based on our tissue bank of aldosterone-producing adenomas (APA), we identified a novel somatic ATP2B3 K416_F418delinsN mutation. The affected individual was a 53 year-old man with a 4 year history of hypertension. Computed tomography (CT) showed bilateral adrenal masses of 1.6 (left) and 0.5 cm (right) in size. An adrenal venous sampling (AVS) showed a lateralization index (LI) of 2.2 and a contralateral suppression index (CLS) of 0.12; indicating left functional predominance. After a left unilateral adrenalectomy, he achieved partial biochemical and hypertension–remission. This classical adenoma harbored a novel ATP2B3 K416_F418delinsN somatic mutation, which is a deletion from nucleotides 1248 to 1253. The translated amino acid sequence from 416 to 418, reading as lysine-phenylalanine-phenylalanine, was deleted; however, an asparagine was inserted due to merging of residual nucleotide sequences. The CYP11B2 immunohistochemistry staining demonstrated strong immunoreactivity in this classical adenoma. The ATP2B3 K416_F418delinsN mutation is a functional mutation in APA, since HAC15 cells, a human adrenal cell line, transfected with the mutant gene showed increased CYP11B2 expression and aldosterone production.

Aldosterone and Primary Aldosteronism: Star Performers in Hypertension Research

Aldosterone and Primary Aldosteronism: Star Performers in Hypertension Research

Hypertension Editors' Picks: Hyperaldosteronism.

Hypertension Editors' Picks: Hyperaldosteronism.

International Histopathology Consensus for Unilateral Primary Aldosteronism.

Develop a consensus for the nomenclature and definition of adrenal histopathologic features in unilateral primary aldosteronism (PA). Unilateral PA is the most common surgically treated form of hypertension. Morphologic examination combined with CYP11B2 (aldosterone synthase) immunostaining reveals diverse histopathologic features of lesions in the resected adrenals. Surgically removed adrenals (n = 37) from 90 patients operated from 2015 to 2018 in Munich, Germany, were selected to represent the broad histologic spectrum of unilateral PA. Five pathologists (Group 1 from Germany, Italy, and Japan) evaluated the histopathology of hematoxylin-eosin (HE) and CYP11B2 immunostained sections, and a consensus was established to define the identifiable features. The consensus was subsequently used by 6 additional pathologists (Group 2 from Australia, Brazil, Canada, Japan, United Kingdom, United States) for the assessment of all adrenals with disagreement for histopathologic diagnoses among group 1 pathologists. Consensus was achieved to define histopathologic features associated with PA. Use of CYP11B2 immunostaining resulted in a change of the original HE morphology-driven diagnosis in 5 (14%) of 37 cases. Using the consensus criteria, group 2 pathologists agreed for the evaluation of 11 of the 12 cases of disagreement among group 1 pathologists. The HISTALDO (histopathology of primary aldosteronism) consensus is useful to standardize nomenclature and achieve consistency among pathologists for the histopathologic diagnosis of unilateral PA. CYP11B2 immunohistochemistry should be incorporated into the routine clinical diagnostic workup to localize the likely source of aldosterone production.

SAT-549 Identification of Somatic Mutations in CLCN2 as a Cause of Aldosterone-Producing Adenomas

Background: Primary aldosteronism (PA) results from both unilateral and bilateral adrenal disease. Unilateral disease is most often caused by aldosterone-producing adenomas (APAs). We recently identified aldosterone-driver somatic mutations in approximately 90% of APAs using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided DNA sequencing approach. In the present study, we analyzed DNA from APA samples found to be mutation negative. Methods: Formalin-fixed paraffin-embedded tissue samples from PA patients who underwent adrenalectomy were studied. Genomic DNA was isolated from 118 APAs (identified by CYP11B2 IHC). Next generation sequencing (NGS) was performed to identify known aldosterone-driver mutations in KCNJ5, ATP1A1, ATP2B3, and CACNA1D. APA DNA that was mutation negative and the adjacent normal adrenal tissue DNA were subjected to Whole Exome Sequencing (WES). Results: Targeted NGS and WES detected two variants in the voltage-gated chloride channel ClC-2 (encoded by CLCN2), which were confirmed by Sanger sequencing. One of the CLCN2 mutations (p.Gly24Asp) was identical to that previously found to cause germline early-onset PA. The second CLCN2 mutation, which would affect the same region of the protein, was an unreported PA mutation (p.Met22fs). The presence of these variants in two tumors suggests that CLCN2 mutations as a cause of APAs are rare with an approximate prevalence of 1.7% (2/118 APAs). Conclusion: In this study, we identified somatic mutations in CLCN2, in two of 118 APAs. Germline variants in this gene have previously been shown to cause of familial hyperaldosteronism type II and the current findings indicate that similar mutations cause a small proportion of APAs. These findings also indicate that WES of CYP11B2-guided mutation negative APAs can help determine rarer genetic causes of sporadic PA.

SAT-554 Genetic Profile of Early-Onset Aldosterone-Producing Adenomas

Background: Aldosterone-producing adenoma (APA) is a major subtype of primary aldosteronism (PA) which is the most common cause of endocrine-related hypertension. The Endocrine Society clinical practice guideline suggests that young patients (< 35 years old) with a CT-detected adrenocortical adenoma and typical phenotype of PA may not need adrenal venous sampling prior to adrenalectomy. In recent years, aldosterone-driver somatic mutations have been identified in APA, and prevalence studies suggest potential effects of patient age and sex. However, the rare nature of early-onset PA has prevented a detailed study of the histologic characteristics and aldosterone-driver somatic mutations in adrenal tumors from these patients. Objective: To determine histologic and somatic mutation profile in early-onset APA. Methods: Fifty-five formalin-fixed paraffin-embedded (FFPE) adrenals from patients at the age of 35 years old or younger who underwent adrenalectomy at the participating centers were studied (45 women, 9 men, and 1 unknown sex). CYP11B2 immunohistochemistry (IHC)-guided tumor capturing was used to selectively obtain DNA from APA. Mutation status was determined either by Sanger sequencing or targeted next-generation sequencing. Results: CYP11B2 IHC identified APAs in all adrenal specimens. Solitary APAs were found in 53 adrenals. One adrenal had multiple APAs and one had a dominant CYP11B2-negative tumor and a smaller APA. In total, DNA from 57 APAs were sequenced. Two APAs were excluded from the analysis due to low sample quality. In 52 of the 55 APAs, somatic mutations were identified in one of the aldosterone-driver genes or CTNNB1 gene, encoding β-catenin. The most common genetic alteration was seen in KCNJ5 (37/55, 67%), followed by CACNA1D (7/55, 13%), ATP1A1 (3/55, 5%), CTNNB1 (3/55, 5%), and ATP2B3 (2/55, 4%). No sex difference in the prevalence of KCNJ5 mutation was observed in this age group. Conclusion: The majority of adrenals from early-onset PA patients had a solitary APA. Regardless of sex, the most common genetic cause of early-onset APA was somatic mutations in KCNJ5.

SAT-555 Can Histology Predict the Presence of KCNJ5 Somatic Mutation in Aldosterone-Producing Adenomas?

Aldosterone-producing adenoma (APA) is well known to harbor marked intratumoral heterogeneity in terms of morphology and CYP11B2 (aldosterone synthase) localization. In histology, APA is generally characterized by two distinct cell subtypes, namely “clear cells” and “compact cells”. Clear tumor cells harbor abundant lipid droplets in their cytoplasms and compact tumor cells generally featuring small round shape have abundant intracytoplasmic organelles including mitochondria.Relatively close correlation between these histological characteristics (morphology and CYP11B2 immunohistochemistry) and genotypes of aldosterone-driver gene somatic mutation has been reported. Among them, KCNJ5-mutated APAs have been reported to harbor clear cell predominant features, while APAs with other rare somatic mutations including ATP1A1, ATP2B3 and CACNA1D harbor heterogenous or relatively compact cell predominant morphometry. However, these previous evaluation were based on eyeball analysis with relatively low reproducibility. Therefore, we developed the more quantitative methods using digital image software in order to analyze the widespread area, which can reflect intratumoral heterogeneity, with high reproducibility to analyze the further detailed correlation between histopathological characteristics and genotype in APA. We explored the utility of immunohistochemistry including CYP11B2 and KCNJ5. We further attempted to propose histopathological scoring system to predict the presence of KCNJ5 somatic mutation in APAs.Results of our present study revealed that KCNJ5 was predominantly immunolocalized in zona glomerulosa among adrenal cortex (vs. ZF, P=0.0002, vs. ZR, P=0.0002), furthermore, predominantly in APCCs than in non-APCCs (P=0.0019). Among the tumors, KCNJ5 immunoreactivity was significantly higher in KCNJ5-wild type APAs than in mutated ones (P=0.0037). KCNJ5-mutated APAs had significantly lower nuclear / cytoplasm ratio and abundant clear cell components than those with wild type, harboring large tumor size. In conclusion, we firstly proposed a novel histopathological predicting scoring system for the presence of KCNJ5 somatic mutation, including the following histopathological findings; N/C ratio, clear cell (%), tumor size, CYP11B2 immunoreactivity and KCNJ5 immunoreactivity. It is true that no single histological factors above could precisely predict the presence of KCNJ5 somatic mutation but this newly developed combined histopathological predicting scoring system could provide relatively high accuracy to predict KCNJ5 somatic mutation in APAs (AUC=96%, sensitivity:100%, specificity:90%, 4 points or more). However, further prospective validation by large number of cases is required for clarification.

OR19-02 Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Protein Expression in Adrenocortical Progenitor Cells, Aldosterone Producing Cell Clusters and Adrenal Adenomas Derived from Postmenopausal Women

Objective/BackgroundAdrenal pathologies are more common in women than men. Embryologically the adrenals and gonads develop from the adrenogenital ridge with differential migration and differentiation. We hypothesized that in adult females there are adrenocortical progenitor cells that express the LH/hCG-R and proliferate in response to elevated LH. Indeed, several case reports demonstrated LH/hCG-R expression in adrenal secretory tumors in postmenopausal and pregnant females. In aging adults, nests of cells known as aldosterone-producing cell clusters (APCCs) that may be precursors to aldosterone producing adenomas are frequently detected. We retrospectively studied the immunohistochemical expression of LH/hCG-R in normal adrenals, adrenal adenomas and APCCs in archival specimens derived from post-menopausal women.MethodsArchival specimens from adrenal adenomas derived from 23 women >55 years of age were examined. Clinical data was obtained in a blinded fashion and hormonal data was available in 9/23 cases; 6/9 were secreting cortisol and 3/9 adenomas were secreting aldosterone. In addition, 6 samples derived from a repository of normal adrenal tissues from deceased kidney donors (1 male, and 5 postmenopausal females) were studied. All specimens were immunostained for LH/hCG-R and the adrenal stem cell marker DLK1 that facilitates the maintenance of an undifferentiated phenotype. The normal adrenal tissues were also stained for aldosterone synthase (CYP11B2) to detect APCCs. The slides were reviewed and graded by a pathologist in a blinded fashion.ResultsExpression of LH/hCG-R was demonstrated in both normal and adenomatous tissues in all 23 specimens. The staining in adenomas was heterogeneous, with clusters of densely stained LH/hCG-R positive cells in all specimens. There were less densely stained clusters in normal adjacent adrenocortical tissue that was most prominent in the subcapsular, zona glomerulosa region, an area where the putative adrenal cortical stem cells are found as well as the zona reticularis. Double staining for the stem cell marker DLK1 and LH/hCG-R confirmed that these cells represent adrenocortical progenitor cells. CYP 11B2 immunohistochemistry of normal adrenals demonstrated cell foci dipping from the capsule into the zona fasciculata classified as APCCs that co-expressed cytoplasmic LH/hCGR.ConclusionAdrenal adenomas and APCCs derived from postmenopausal women exhibited heterogeneous but strong immunohistochemical expression of LH/hCG-R in all samples. Interestingly, DLK1-positive adrenocortical stem cells in the subcapsular zone also expressed LH/hCG-R. These data may provide insights into the female predominance of adrenal pathologies, particularly in postmenopausal women with high LH levels. The LH/hCG-R may be a viable target for treatment of adrenal adenomas in postmenopausal women.

SUN-195 Bilateral Aldosterone-Producing Adenomas: A New Subtype of Bilateral Primary Aldosteronism?

Background: Primary aldosteronism (PA) is the most common cause of endocrine hypertension (HT). PA subtypes include aldosterone-producing adenomas (APA) and bilateral adrenal hyperplasia. To date, few PA patients with bilateral adenomas have been reported, but only one case was well characterized by anatomopathological analysis and clinical outcome after adrenal sparing surgery (1). Clinical case: A 53-year-old woman was referred to investigate resistant HT and hypokalemia. (3.0 mEq/L). PA screening revealed aldosterone (A) of 37.9 ng/dL, renin (R) < 1.6 (4.4-46.1 mUI/L), A/R ratio of 24.8. Confirmatory testing confirmed PA diagnosis: seated saline infusion test (A= 83.3 ng/dL) and intravenous furosemide test (R= 3.1 mUI/L; positive test <13 mUI/L). Hypercortisolism investigation revealed a non-suppressible cortisol after an overnight 1 mg low-dose dexamethasone suppression [cortisol (C)= 2.9 μg/dL and dexamethasone= 701 (˃130 ng/dL)], and normal urinary free cortisol, midnight salivary cortisol, plasma DHEAS and ACTH levels. Computed tomography demonstrated bilateral adrenal nodules without adrenal thickening: 3.5 cm right nodule (pre-contrast density of 7UH density; absolute wash-out of 71%) and 2.5 cm left nodule (pre-contrast density of 8UH density; absolute wash-out of 78%). Sequential adrenal venous (AV) sampling (AVS) under continuous cosyntropin infusion showed a lateralization index of 3.4 (bilateral disease <4). Then, the patient underwent right adrenalectomy and left nodulectomy. In the postoperative period, she presented normalization of K+ levels and complete HT remission. She remained under hydrocortisone replacement for 2 months. After 2 months, biochemical evaluation revealed normal basal cortisol levels (13.3 µg/dL) and biochemical cure of PA (A= 3.1 ng/dL and R= 15.3 mUI/L). Currently, she doesn’t have symptoms of adrenal insufficiency after discontinuation of hydrocortisone. Anatomopathological analysis showed bilateral adenomas (Weiss score of 0) in both sides without adjacent hyperplasia. CYP11B2 immunohistochemistry displayed a strong staining in 50% of cells in the right adenoma and in 30% of cells in the left adenoma. Few aldosterone-producing cell clusters (APCC) were identified in the right zona glomerulosa, which is a frequent finding in normal adrenals. Conclusion: We herein described a very rare case of PA caused by bilateral-producing adenomas, confirmed by AVS and CYP11B2 staining after adrenal sparing surgery.

The Potential Role of Aldosterone-Producing Cell Clusters in Adrenal Disease.

Primary aldosteronism (PA) is the most common cause of secondary hypertension. The hallmark of PA is adrenal production of aldosterone under suppressed renin conditions. PA subtypes include adrenal unilateral and bilateral hyperaldosteronism. Considerable progress has been made in defining the role for somatic gene mutations in aldosterone-producing adenomas (APA) as the primary cause of unilateral PA. This includes the use of next-generation sequencing (NGS) to define recurrent somatic mutations in APA that disrupt calcium signaling, increase aldosterone synthase (CYP11B2) expression, and aldosterone production. The use of CYP11B2 immunohistochemistry on adrenal glands from normal subjects, patients with unilateral and bilateral PA has allowed the identification of CYP11B2-positive cell foci, termed aldosterone-producing cell clusters (APCC). APCC lie beneath the adrenal capsule and like APA, many APCC harbor somatic gene mutations known to increase aldosterone production. These findings suggest that APCC may play a role in pathologic progression of PA. Herein, we provide an update on recent research directed at characterizing APCC and also discuss the unanswered questions related to the role of APCC in PA.

Genetic, Cellular, and Molecular Heterogeneity in Adrenals With Aldosterone-Producing Adenoma.

Aldosterone-producing adenoma (APA) cause primary aldosteronism-the most frequent form of secondary hypertension. Somatic mutations in genes coding for ion channels and ATPases are found in APA and in aldosterone-producing cell clusters. We investigated the genetic, cellular, and molecular heterogeneity of different aldosterone-producing structures in adrenals with APA, to get insight into the mechanisms driving their development and to investigate their clinical and biochemical correlates. Genetic analysis of APA, aldosterone-producing cell clusters, and secondary nodules was performed in adrenal tissues from 49 patients by next-generation sequencing following CYP11B2 immunohistochemistry. Results were correlated with clinical and biochemical characteristics of patients, steroid profiles, and histological features of the tumor and adjacent adrenal cortex. Somatic mutations were identified in 93.75% of APAs. Adenoma carrying KCNJ5 mutations had more clear cells and cells expressing CYP11B1, and fewer cells expressing CYP11B2 or activated β-catenin, compared with other mutational groups. 18-hydroxycortisol and 18-oxocortisol were higher in patients carrying KCNJ5 mutations and correlated with histological features of adenoma; however, mutational status could not be predicted using steroid profiling. Heterogeneous CYP11B2 expression in KCNJ5-mutated adenoma was not associated with genetic heterogeneity. Different mutations were identified in secondary nodules expressing aldosterone synthase and in independent aldosterone-producing cell clusters from adrenals with adenoma; known KCNJ5 mutations were identified in 5 aldosterone-producing cell clusters. Genetic heterogeneity in different aldosterone-producing structures in the same adrenal suggests complex mechanisms underlying APA development.

Mass Spectrometry Imaging Establishes 2 Distinct Metabolic Phenotypes of Aldosterone-Producing Cell Clusters in Primary Aldosteronism.

Aldosterone-producing adenomas (APAs) are one of the main causes of primary aldosteronism and the most prevalent surgically correctable form of hypertension. Aldosterone-producing cell clusters (APCCs) comprise tight nests of zona glomerulosa cells, strongly positive for CYP11B2 (aldosterone synthase) in immunohistochemistry. APCCs have been suggested as possible precursors of APAs because they frequently carry driver mutations for constitutive aldosterone production, and a few adrenal lesions with histopathologic features of both APCCs and APAs have been identified. Our objective was to investigate the metabolic phenotypes of APCCs (n=27) compared with APAs (n=6) using in situ matrix-assisted laser desorption/ionization mass spectrometry imaging of formalin-fixed paraffin-embedded adrenals from patients with unilateral primary aldosteronism. Specific distribution patterns of metabolites were associated with APCCs and classified 2 separate APCC subgroups (subgroups 1 and 2) indistinguishable by CYP11B2 immunohistochemistry. Metabolic profiles of APCCs in subgroup 1 were tightly clustered and distinct from subgroup 2 and APAs. Multiple APCCs from the same adrenal displayed metabolic profiles of the same subgroup. Metabolites of APCC subgroup 2 were highly similar to the APA group and indicated enhanced metabolic pathways favoring cell proliferation compared with APCC subgroup 1. In conclusion, we demonstrate specific subgroups of APCCs with strikingly divergent distribution patterns of metabolites. One subgroup displays a metabolic phenotype convergent with APAs and may represent the progression of APCCs to APAs.

Application of CYP11B2 immunohistochemistry in the histopathological diagnosis of primary aldosteronism

Objective To evaluate the value of CYP11B2 immunohistochemistry in the histopathological diagnosis of primary aldosteronism(PA) among Chinese population. Methods Ninety-six cases of unilateral PA patients were retrospectively enrolled, and with 15 cases of non-hyperfunctioning adenoma, and 15 cases of cortisol producing adenoma as control. CYP11B2 immunohistochemistry was performed in the tumor sections of these patients and was analysed by semi-quantified method. The positive rate of CYP11B2 in tumor specimen of all cases enrolled was calculated and PA patients were subtypely classified based on their histopathological features and CYP11B2 expressions. Results The immune-positive rate of CYP11B2 in the PA group was 91.7% (88/96), with the control group being 6.7% (2/30, P<0.001). Subgroup analysis revealed that the immune-positive rate of CYP11B2 in the aldosterone producing adenoma group and unilateral hyperplasia group were 91.8% (78/85) and 90.9% (10/11), respectively. Based upon histopathological feature and CYP11B2 immunohistochemistry, PA patients were further subtypely classified as aldosterone producing adenoma (85/96), unilateral diffuse adrenocortical hyperplasia (7/96), unilateral mono-adrenocortical micronodules (1/96), unilateral multiple adrenocortical micronodules (1/96), and unilateral multiple aldosterone-producing cell clusters (multiple APCCs, 2/96). Conclusion CYP11B2 immunohistochemistry may serve as an useful tool for the histopathological diagnosis of PA. Key words: Primary aldosteronism; Immunohistochemistry; Pathological diagnosis