Multifocal, Asymmetric Bilateral Primary Aldosteronism Cannot be Excluded by Strong Adrenal Vein Sampling Lateralization: An International Retrospective Cohort Study

Abstract

Background:

Primary aldosteronism (PA) has been broadly dichotomized into unilateral and bilateral forms. Adrenal vein sampling (AVS) lateralization indices (LI) ≥2-4 are the standard-of-care to recommend unilateral adrenalectomy for presumed unilateral PA.

Objective:

To assess the rates and characteristics of residual PA after AVS-guided adrenalectomy.

Design:

Retrospective, cohort study.

Setting:

7 referral centers from 4 continents.

Participants:

Patients with PA who underwent unilateral adrenalectomy based on AVS LI≥4.

Measurements:

The proportion of patients without PA cure. Aldosterone synthase (CYP11B2) immunohistochemistry and next generation sequencing were performed on available formalin-fixed paraffin-embedded adrenal tissue.

Results:

The cohort included 283 patients who underwent AVS-guided adrenalectomy, followed for a median of 326 days postoperatively. Lack of PA cure was observed in 16% of consecutive patients, and in 22 patients with lateralized PA on both baseline and cosyntropin-stimulated AVS. Among patients whith residual PA post-operatively, 73% had multiple CYP11B2 positive areas within the resected adrenal tissue (vs. 23% in those cured), wherein CACNA1D mutations were most prevalent (63% vs. 33% in those cured). In adjusted regression models, independent predictors of post-operative residual PA included Black vs. White race (OR 5.10, 95% CI 1.45-17.86), AVS lateralization only at baseline (OR 8.93, CI 3.00-26.32 vs. both at baseline and after cosyntropin stimulation), and CT-AVS disagreement (OR 2.75, CI, 1.20-6.31).

Conclusion:

Multifocal, asymmetric bilateral PA is relatively common, and it cannot be excluded by robust AVS lateralization. Long-term postoperative monitoring should be routinely pursued, to identify residual PA and afford timely initiation of targeted medical therapy.

Graphical Abstract

Introduction

Primary aldosteronism (PA) is a common cause of treatment-resistant hypertension.¹ PA enhances the risk of cardiovascular and renal morbidity above that conferred by hypertension,^2–8 via direct target organ insults, with resultant inflammation, fibrosis, and necrosis.^{9, 10} Timely recognition and targeted therapy of PA is critical to mitigate its clinical complications.^{2, 11–14}

Personalized therapy of PA therapy takes into account two broad subtypes: unilateral PA, commonly attributed to surgically curable aldosterone-producing adenomas (APA), and bilateral PA (BPA), treated with mineralocorticoid receptor antagonists.¹⁵ Overall, unilateral adrenalectomy is considered more effective in improving blood pressure control,¹⁶ and in reducing the risk of incident cardiovascular events,¹⁴ atrial fibrillation,¹⁷ and renal insufficiency¹³ than medical therapy for PA.

Adrenal vein sampling (AVS) is the current standard-of-care for identifying patients with lateralized PA, who are commonly referred for unilateral adrenalectomy.¹⁸ Substantial heterogeneity in AVS procedural protocols and data interpretation exists even among referral centers,¹⁹ and these differences impact the recommendation for or against surgery.²⁰ Data on long-term postoperative follow up are limited, and include variability in PA subtyping tests and criteria used.^{21, 22} Nevertheless, because AVS relies on comparative adrenal aldosterone production, with limited applicability of absolute hormonal concentrations within each adrenal vein, we hypothesized that even robust aldosterone lateralization cannot exclude bilateral PA. This study was designed to assess the rates and characteristics of PA cases that lack biochemical cure after unilateral adrenalectomy, despite being guided by strong AVS lateralization.

Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request, and after data sharing agreements, as required per each participating institution.

Study Design

We conducted an international, retrospective, cohort study at 7 referral centers from 4 continents, 6 of which are members of the American-Australian-Asian Adrenal Alliance (A5) international research consortium. Studies were led with approval from each institutional review board. Patients provided written informed consent for use of biological samples. Medical records review was conducted with waiver for informed consent. All data shared outside of the originating institution were de-identified.

Participants

We included all consecutive patients with confirmed PA who underwent AVS-guided unilateral adrenalectomy between Jan 2016 and July 2022 at three institutions where AVS is performed with and without cosyntropin stimulation: University of Michigan, U.S., Tohoku University, Japan, and Monash Health, Australia. In addition, to broaden the histopathology and aldosterone-driver mutation studies, we included patients from 4 other centers (Munich University, University of Pennsylvania, Brigham and Women’s Hospital, and Centre hospitalier de l’Université of de Montréal) who did not experience biochemical cure following AVS-guided adrenalectomy, and who had adrenal tissue available (Figure 1). We excluded patients who had no post-operative hormonal evaluation.

Figure 1.

Study flow

BWH, Brigham and Women’s Hospital; UPenn, University of Pennsylvania; Aldo, aldosterone; ARR, aldosterone to renin ratio.

Procedures

All patients underwent AVS following the institutional protocol. AVS was conducted both prior to and after administration of cosyntropin at University of Michigan,²³ Tohoku University,²⁴ Monash Health, Centre hospitalier de l’Université de Montréal,²⁵ and Brigham and Women’s Hospital,²⁶ as described previously. At University of Munich, AVS was conducted only without cosyntropin,²⁷ and at University of Pennsylvania²⁸ only after cosyntropin stimulation. Datasets where the selectivity index (calculated as a ratio of cortisol at the catheter site vs. peripheral circulation) was < 2 at baseline and <5 after cosyntropin stimulation were excluded. LI was calculated as a ratio of [aldosterone/cortisol] between the two adrenal veins, with the lowest of the two as denominator. A LI≥4 was considered lateralized (L), and a LI<4 was considered bilateral (B) PA. We excluded patients with all LI<4. In patients who underwent AVS both before and after cosyntropin stimulation, both results were recorded, rendering three subgroups: lateralization only at baseline (LB), lateralization only after cosyntropin stimulation (BL), or consistent lateralization (LL). Contralateral suppression index (CSI) was calculated based as a ratio of [aldosterone/cortisol] between the non-dominant adrenal vein and peripheral circulation.

Plasma aldosterone concentration (PAC), renin (plasma renin activity, PRA, or direct renin concentration, DRC), and basic metabolic panels were measured at local laboratories. Suppressed renin was defined as PRA <1 ng/mL/hour or equivalent DRC at each institution. A conversion factor of 8:1 to was used to transform DRC to PRA equivalent values for group comparisons.

Cross-sectional abdominal imaging radiology reports were used to ascertain agreement with AVS results. Unilateral adrenal nodules and/or thickening (as determined by the local radiologists) coinciding with the side of AVS lateralization was considered agreement. Bilateral adrenal abnormalities, normal adrenal glands, or structural abnormalities on the side opposite to AVS lateralization were considered discordant with AVS.

In all patients where formalin fixed paraffin embedded (FFPE) adrenal tissue was available, we performed immunohistochemistry (IHC) for aldosterone synthase (CYP11B2), to map out the source(s) of aldosterone excess, as described previously.²⁹ CYP11B2-expressing lesions were selectively captured and DNA isolated.

For mutation analysis, multiplexed PCR–based next generation sequencing (NGS) was conducted using Ion Torrent Ampliseq sequencing (Thermo Fisher Scientific), as previously described.³⁰ The panel for library preparation included amplicons targeting the full coding regions of known aldosterone-driving genes, including the most frequently affected: KCNJ5, CACNA1D, CACNA1H, CLCN2, CTNNB1, ATP1A1, and ATP2B3. In most cases with multiple CYP11B2 areas, NGS was conducted on DNA isolated from the dominant lesion.

Outcomes

Postoperative biochemical outcomes were assessed based on modified PASO criteria,³¹ as follows: complete PA cure, if PAC<10 ng/dL or aldosterone-to-renin ratio (ARR) <30 (ng/dL)/(ng/mL/hour); partial PA improvement, if PAC fell by >50%, but PAC >10 ng/dL and ARR >30 (ng/dL)/(ng/mL/hour); and persistent PA if PAC fell by <50%, PAC >10 ng/dL and ARR >30 (ng/dL)/(ng/mL/hour). In addition, we used the PASO hypokalemia criteria, as well as clinical criteria.³¹ Screening hormonal data were used as baseline. The latest available post-operative follow-up data, or the set prior to initiation of mineralocorticoid receptor antagonists where applicable, were used for analysis.

Statistical analysis

We report clinical characteristics as frequencies for categorical variables, and median and interquartile range [IQR] for continuous variables. Comparison of continuous variables between two or three independent groups were conducted using the Mann-Whitney U test, or Kruskal-Wallis test, respectively. The Chi-squared test and Fisher’s exact test were used for comparison of proportions between groups. Univariate and multivariable logistic regression were conducted to assess the association between postsurgical outcomes as dependent variable, and clinical variables as predictors. Adjusted logistic regression models were based on clinical relevance, collinearity, and interactions. Data analysis was conducted using SAS 9.4 (North Carolina State University) and Prism (GraphPad Software, Inc., Boston, MA). Statistical significance was accepted at two-tailed p < 0.05.

Results

Over the study period, 310 consecutive patients underwent AVS-guided adrenalectomy at the three institutions with complete datasets (Figure 1). Of these, 48 patients were excluded due to lack of biochemical follow up and/or having all LI<4. Among the 262 consecutive patients with post-operative data available from three centers, 42 (16%) patients had evidence of residual PA following surgery, including 29 (11%) with partial PA improvement, and 13 (5%) without biochemical improvement. From the 4 additional centers, review of available cases led to the identification of 21 eligible patients who lacked PA cure following adrenalectomy guided by a LI≥4 on AVS. This group was similar in age, gender, BMI, and AVS results with the 42 patients without PA cure from the consecutive cohorts (Table S1).

The final cohort included a total of 283 patients, with a mean age of 52.6 years (range: 19-78 years), of whom 174 (61.5%) were men, 103 (36.4%) were White, 100 (35.3%) Asian, 28 (9.9%) Black, and 52 (18.4%) of other or unknown race (Table 1). Patients were followed for a median of 326 [82-603] days postoperatively. Post-operative clinical outcomes were available in 270 patients (Table S2). Expectedly, all patients with post-operative biochemical cure had either resolution of hypertension (34%) or partial clinical improvement (66%). Of patients without post-operative PA cure, 19% had no clinical benefit, 73% had partial clinical benefit, and 8% had hypertension resolution.

Table 1.

Characteristics of study participants.

Parameters	All (283)	PA Cure (220)	No Cure (63)	p value
Age (years)	53 (19-78)	53 (19-78)	54 (19-75)	0.33
Gender (Men)	174 (61.5%)	137 (62.3%)	37 (58.7%)	0.61
Race White	103 (36.4%)	85 (38.6%)	18 (28.6%)	0.0003
Asian	100 (35.3%)	85 (38.6%)	15 (23.8%)
Black	28 (9.9%)	14 (6.4%)	14 (22.2%)
Other/Unknown	52 (18.4%)	36 (16.4%)	16 (25.4%)
BMI (kg/m2)	28.7 [24.3-33.9]	28.3 [24.4-33.6]	30.7 [24.2-35.0]	0.32
Creatinine (mg/dL)	0.9 [0.7-1.1]	0.9 [0.7-1.1]	0.9 [0.7-1.2]	0.08
Serum [K+] (mmol/L)	3.7 [3.3-4.0]	3.7 [3.3-4.0]	3.8 [3.4-4.1]	0.45
Baseline PAC (ng/dL)	33.2 [23.7-53.9]	33.8 [23.8-56.5]	31.6 [23.7-45.7]	0.24
Baseline PRA (ng/mL/h)	0.3 [0.2-0.6]	0.3 [0.2-0.6]	0.3 [0.2-0.6]	0.81
Baseline ARR [(ng/dL)/(ng/mL/h)]	124.0 [68.1-237.5]	125.7 [66.1-248.8]	118.5 [73.3-228.5]	0.67
LI Pre-ACTH	9.9 [4.8-23.0]	11.3 [4.9-27.8]	7.2 [4.6-16.2]	0.07
LI Post-ACTH	11.4 [5.5-33.3]	17.0 [7.2-41.9]	4.9 [2.5-7.6]	<0.0001
CSI Pre-ACTH	0.5 [0.2-1.0]	0.41 [0.2-0.8]	0.9 [0.5-1.5]	<0.0001
CSI Post-ACTH	0.2 [0.1-0.5]	0.2 [0.1-0.4]	0.7 [0.4-1.3]	<0.0001

PAC, plasma aldosterone concentration; PRA, plasma renin activity; ARR, aldosterone-to-renin ratio; LI, lateralization index; CSI, contralateral suppression index.

Overall, compared to patients with biochemical cure, those without biochemical cure were more frequently Black (22.2% vs. 6.4%), and had lower LI, particularly when AVS was performed with cosyntropin stimulation (LI: 4.8 [2.5-7.6] vs. 17.0 [7.2-41.9], p<0.0001, Table 1). Conversely, patients with complete PA resolution had more profound contralateral suppression, both at baseline (CSI: 0.4 [0.1-0.8] vs. 0.9 [0.5-1.5], p<0.0001) and after cosyntropin stimulation (0.2 [0.1-0.4] vs. 0.7 [0.4-1.3], p<0.0001, Table 1).

As a group, patients with partial PA improvement had the highest PAC and ARR (Table S3). A gradient of LI was noted across the groups, being highest in patients with PA cure, and lowest in those with no biochemical benefit from surgery (Table S1). The group with absent surgical benefit had the lowest proportion of patients with consistent AVS lateralization pre- and post-cosyntropin, and the highest proportion of patients with AVS lateralization exclusively at baseline (Table 2).

Table 2.

Comparison of AVS, cross-sectional imaging, and aldosterone driver mutations of study participants, stratified by post-operative biochemical outcomes.

	PA cure (220)	Partial improvement (34)	No improvement (29)	p value
AVS Lateralization a
LL	161 (73,2%)	15 (46.9%)	7 (33.3%)	<0.0001
LB	11 (5.0%)	9 (28.1%)	11 (52.4%)
BL	36 (16.4%)	4 (12.5%)	3 (14.4%)
FL	12 (5.5%)	4 (12.5%)	0 (0 %)
CT findings b
Ipsilateral nodule	165 (79.7%)	20 (58.8%)	11 (37.9%)	<0.0001
Contralateral nodule	7 (3.4%)	1 (2.9%)	3 (10.3%)
Bilateral	9 (4.3%)	2 (5.9%)	6 (20.7%)
No nodule	26 (12.6%)	11 (32.4%)	9 (31.0%)
Aldosterone-driver mutation c
CACNA1D	41 (33.1%)	10 (58.8%)	9 (69.2%)	0.0035
KCNJ5	52 (41.9%)	5 (29.4%)	3 (23.1%)
ATPase	31 (25.0%)	2 (11.8%)	1 (7.7%)

^aAVS results were classified as lateralized (L) or bilateral (B) based on lateralization indices ≥4 or <4, respectively. Baseline (first letter) and cosyntropin-stimulated (second letter) AVS results are shown. F stands for failed adrenal vein cannulation. Patients from Munich University (7) had AVS only without cosyntropin stimulation, and patients from University of Pennsylvania (3) had AVS only without cosyntropin stimulation, and they were not included in this table.

^bCross-sectional imaging data was not available in 13 patients with PA cure.

^cIn cases with multiple CYP11B2-positive areas, DNA sequencing was performed on the dominant lesions.

Within the subgroup of 183 patients with LI≥4 both pre- and post-cosyntropin stimulation, 22 patients had lack of PA cure. Compared with those with curative adrenalectomy, patients with residual PA were older (58 vs. 51 years, p=0.01), and, although they had similar PAC and ARR, patients with residual PA had lower LI and higher CSI (Table 3).

Table 3.

Comparison of patients with and without PA cure in whom AVS showed aldosterone lateralization both before and after cosyntropin stimulation.

Parameter	Cure (161)	No Cure (22)	p value
Age (Years)	51 [43-60]	58 [51-66]	0.01
Gender (Men)	97 (60.3%)	15 (68.2%)	0.64
Race White	59 (47.7%)	10 (47.6%)	0.30
Black	12 (9.1%)	4 (19.0%)
Asian	61 (46.2%)	7 (33.3%)
BMI (kg/m2)	28.5 [23.8-34.1]	29.7 [24.2-34.3]	0.71
Creatinine (mg/dL)	0.9 [0.7-1.0]	0.9 [0.7-1.2]	0.36
Serum [K+] (mmol/L)	3.7 [3.3-3.9]	3.8 [3.4-3.9]	0.93
Baseline PAC (ng/dL)	36.3 [24.0-58.9]	36.6 [28.5-50.2]	0.88
Baseline PRA (ng/mL/h	0.3 [0.2-0.6]	0.3 [0.2-0.5]	0.61
Baseline ARR [(ng/dL)/(ng/mL/h)]	125.7 [69.9-262.3]	109.7 [72.5-246.2]	0.84
LI Pre-ACTH	17.4 [8.9-39.9]	8.8 [6.3-25.9]	0.03
LI Post-ACTH	21.2 [9.6-51.5]	7.5 [5.3-14.2]	<0.0001
CSI Pre-ACTH	0.4 [0.2-0.7]	1.1 [0.4-1.5]	0.0007
CSI Post-ACTH	0.1 [0.1-0.3]	0.5 [0.4-0.8]	<0.0001

PAC, plasma aldosterone concentration; PRA, plasma renin activity; ARR, aldosterone-to-renin ratio; LI, lateralization index; CSI, contralateral suppression index.

Agreement between AVS and imaging studies

Of patients without postoperative improvement of PA, only 38% had concordant AVS lateralization and cross-sectional imaging findings, as compared to 59% of patients with partial PA improvement, and 80% of patients with PA cure (Table 2). Patients with absent PA improvement following surgery had the highest rates of normal adrenals (31%) or bilateral nodules (21%) on cross sectional imaging.

CYP11B2 expression

Of 63 patients without biochemical cure, tissue was available for CYP11B2 IHC in 55 (87%) patients. Multiple CYP11B2 positive areas were identified in the available blocks in 40 (73%) patients. Representative images illustrating various combinations of aldosterone-producing nodules or micronodules, with or without dominant APAs, non-functional adenomas, or diffuse hyperplasia are shown on Figure 2. Conversely, of 90 available adrenal glands removed from patients with complete PA cure, 69 (77%) harbored a single CYP11B2 structure (66 an APA and 3 an aldosterone-producing nodule). Of the remaining 21 (23%) adrenals, 18 had a dominant APA and either 1-2 punctiform aldosterone-producing micronodules (13) or an aldosterone-producing nodule (5).

Figure 2.

Illustrative images of CYP11B2 immunohistochemistry in adrenals resected from patients without post-operative PA cure.

Multiple CYP11B2 areas were detected in 73% of patients who lacked post-operative biochemical cure, in various combinations of shapes and sizes, such as aldosterone-producing micronodules (A and E), aldosterone-producing nodules (B, D, E, F), and/or aldosterone-producing adenomas (C and F), with or without non-functioning adrenal adenomas (C and E).

Aldosterone driver mutations

In total, aldosterone-driver mutations were identified by NGS in 154 patients with tissue available, including 30 patients without biochemical PA cure following AVS-guided adrenalectomy. Among patients without biochemical cure, CACNA1D mutations were most prevalent (63%), followed by KCNJ5 mutations (27%), and ATP1A1 mutations (10%). In comparison, within the group with surgical resolution of PA, the most prevalent aldosterone-driver mutation was KCNJ5, which accounted for 42% of cases, while CACNA1D mutations were found in 33% of patients (Table 2).

Clinical predictors of biochemical cure with unilateral adrenalectomy

Univariate logistic regression showed that the odds of postsurgical PA cure (presented as odds ratio, OR and 95% confidence interval, CI) were 3.6-fold higher in patients with CT-AVS lateralization agreement (CI, 1.9-7.0). In addition, the odds of biochemical cure increased with a higher LI on cosyntropin-stimulated AVS (OR, 1.2; CI, 1.1-1.3), and with a lower CSI, particularly after cosyntropin stimulation (Figure 3A). Conversely, the odds of surgical cure were 4.2-fold lower in Black vs. White patients (CI, 1.5-11.6), and 13.3-fold lower in those with exclusive baseline lateralization vs. those with consistent AVS lateralization (CI, 5.6-31.2).

Figure 3.

Factors associated with post-operative biochemical cure.

A. Univariate logistic regression. B. Multivariable logistic regression (203 patients were included in the model).

LI, lateralization index; CSI, contralateral suppression index; Pre, pre-cosyntropin stimulation Post, post-cosyntropin stimulation; ARR, aldosterone to renin ratio, CT, computed tomography; AVS, adrenal vein sampling; LB, AVS lateralized at baseline, bilateral after cosyntropin stimulation; BL, AVS bilateral at baseline, lateralized after cosyntropin stimulation; LL, AVS lateralized at baseline and after cosyntropin stimulation.

After adjusting for age, sex, and covariates (Figure 3B), the odds of biochemical cure were 5.1-fold lower in Black vs. White patients (CI, 1.5-17.9), 8.9-fold lower in patients with lateralization only with baseline AVS vs. those with consistent AVS lateralization (CI, 3.0-26.3), and 2.8-fold higher in patients with CT-AVS agreement (CI, 1.2-6.3).

Discussion

This study demonstrates that robust AVS lateralization of aldosterone, with or without contralateral suppression, does not exclude the possibility of asymmetrical bilateral PA. The implications of these findings are that unilateral adrenalectomy for lateralizing PA may not always portend cure of PA, as residual PA from the contralateral gland may exist. As such, these findings deconstruct the classical equivalence of lateralized PA with unilateral PA or APA. Rather, we illustrate that lateralized PA can arise from multifocal CYP11B2 areas, at various stages of development, which might be present in one or both adrenal glands.

While isotope-based imaging modalities targeting CYP11B2 are under development,^{32, 33} AVS remains the best functional tool for PA subtyping, and is endorsed by international expert guidelines as the standard-of-care for surgical considerations.^{18, 34–36} Nevertheless, even when renin is undetectable and lateralization is profound, aldosterone is typically measurable in both adrenal veins.³⁷ Considering the high variability in adrenal vein steroid values, absolute adrenal vein aldosterone concentrations are not standardized for determining PA lateralization. Hence, AVS informs only about the comparative aldosterone output of the two adrenal glands, but it cannot exclude asymmetrical bilateral PA. Indeed, previous studies showed that neither LI, nor contralateral suppression are consistent indicators of post-operative PA cure.^37–40 An aldosterone suppression index which eliminates cortisol as a dilution factor has been proposed as a predictor of surgical cure,^{37, 41} but this index has not yet been validated. While contralateral suppression was on average more profound in our group with PA cure, many patients with residual PA displayed contralateral suppression as well. These data suggest that in the context of renin suppression, any aldosterone output from the contralateral adrenal vein might be autonomous.

Absent or partial biochemical improvement following AVS-guided adrenalectomy has been previously reported, but the surgical decision was based on various AVS criteria.^{31, 42} Because AVS lacks harmonization across institutions, treatment recommendations might differ between centers.⁴³ Herein, we included only patients with LI of minimum 4, either on baseline or cosyntropin-stimulated AVS, which has been the most conservative indicator of lateralization,⁴⁴ albeit arbitrary. We found lack of PA cure following surgical removal of adrenal glands that have dramatic AVS lateralization. Intriguingly, however, AVS lateralization exclusively at baseline was most common among patients without biochemical benefit and rare in patients with PA cure. Similarly, data from two Canadian referral centers found that only the post-cosyntropin, but not the baseline LI was associated with biochemical cure.³⁷ We have previously shown that the impact of cosyntropin stimulation on LI is highly variable,^{25, 45} including in patients with consistent lateralization and documented APAs. Additionally, we found that the expression of ACTH receptors (MC2R) varies between APAs with different aldosterone-driver mutations,⁴⁶ which mirrored the LI response to cosyntropin during AVS.³⁸ Taken together, existing evidence suggests that lateralization exclusively at baseline occurs often and it might indicate assymetrical bilateral PA rather than small, curable unilateral APAs.

Aldosterone-producing structures harboring CACNA1D variants have been identified in the adrenal glands of patients with bilateral PA.⁴⁷ Such small aldosterone-producing structures are increasingly prevalent with aging.²⁹ Not surprisingly, CACNA1D were disproportionately represented among patients without biochemical cure, accounting for 59% of cases with partial improvement, and almost 70% of those with absent surgical benefit. Considering that many patients harbored CYP11B2 positive microscopic areas adjacent to APAs, and that only the largest lesions were submitted to NGS, the prevalence of CACNA1D mutations could be even higher. Similarly, CACNA1D somatic mutations were the most frequent genetic abnormality in a study of 10 patients without PA cure from France.⁴² Nevertheless, APA harboring other known aldosterone-driver mutations, with or without small CYP11B2 positive areas were also identified in some patients without PA cure. Cases with histologic and genetic heterogeneity have been previously reported.^{30, 48} Moreover, KCNJ5 mutations were also found in microscopic aldosterone-producing structures.⁴⁸ These findings support the concept of overlapping pathogenesis of APA and bilateral PA, irrespective if symmetrical or asynchronous.

The limitations of cross-sectional imaging in PA subtyping are widely recognized, and they stem in the increasing incidence of non-functional adrenal adenomas with aging,⁴⁹ and the effective aldosterone production from small CYP11B2-positive foci.²⁶ Compared to patients with PA cure, we found lower rates of AVS-CT agreement among patients with absent PA improvement, particularly due to bilateral nodularity or normal adrenal morphology on cross-sectional imaging. In line with these results, we have previously shown that the rate of AVS-imaging disagreement was higher in patients with CACNA1D vs. KCNJ5 mutations, in Black vs. Asian and White individuals, and in those with AVS lateralization only at baseline (vs. those with consistent pre-and post-cosyntropin lateralization).⁵⁰ Indeed, the group lacking PA cure from this cohort was enriched with Black individuals, CACNA1D mutations, and cases with lateralization on baseline AVS only. Other hereditary aspects, such as CYP11B2 variants,⁵¹ renal sodium handling via tubular epithelial sodium channels (ENaC), and predisposition towards PA⁵² might also contribute to lower rates of surgical PA cure in Black individuals.

The inclusion of centers from different continents confers both limitations, due to heterogeneity of diagnostic and subtyping procedures, but also strengths, by providing the largest and most diverse comprehensive study of patients without surgical cure of PA. Postoperative follow up was not standardized, and long-term follow up data was not available in all cases. This reflects the real-world practices in referral centers, and it could lead to under-estimation of recurrent PA. In addition, because a second surgery is not offered to patients without PA cure after unilateral adrenalectomy, confirmatory testing is replaced by pragmatic initiation of mineralocorticoid receptor antagonists. While many antihypertensive agents can interfere with the renin-angiotensin-aldosterone system, most lead to falsely low ARR, by increasing renin. Although beta blockers and direct renin inhibitors lower renin, aldosterone should follow the same trend. Finally, comprehensive tissue analysis for CYP11B2 expression and aldosterone-driver mutations was not possible in all cases; nevertheless, this collection of adrenal tissue stratified by postoperative biochemical outcomes is the largest to date, and our results are in line with data from small studies.

Perspective

This large international cohort study of patients with PA treated with unilateral adrenalectomy following convincing AVS lateralization provides evidence that patients with severe PA and robust lateralization, with or without contralateral suppression, might still have asymmetrical bilateral PA. Furthermore, we show that most cases with lack of PA cure following unilateral adrenalectomy harbor CACNA1D mutations, once again implicating this somatic mutation as the dominant genetic fingerprint of bilateral PA. Black race, discordance between AVS and cross-sectional imaging, and AVS lateralization only at baseline (but not after cosyntropin-stimulation) are additional risk factors for incomplete or absent biochemical benefit following unilateral adrenalectomy. Long-term postoperative follow up is essential to identify patients with residual or recurrent PA, in order to initiate mineralocorticoid receptor antagonist therapy and to reduce the risk of cardiovascular and renal complications.

Novelty and Relevance:

1. What Is New?

   • In a multicenter, international study, we found that 16% of patients with PA that lateralized on AVS did not experience biochemical cure following unilateral adrenalectomy.
   • Of patients without postoperative PA cure, 73% had multiple CYP11B2 positive areas within the resected adrenal tissue.
   • CACNA1D mutations were present in 63% of patients without PA cure following lateralized AVS-guided adrenalectomy (vs. 33% in those cured).
   • Independent risk factors of post-operative residual PA included Black vs. White race, AVS lateralization only at baseline, and CT-AVS disagreement.
2. What Is Relevant?

   • Individuals with PA who are Black, have AVS lateralization only at baseline, but not after cosyntropin stimulation, and discrepant CT-AVS results are at risk for residual PA following AVS-guided adrenalectomy.
3. Clinical/Pathophysiological Implications?

   • Long-term follow up after AVS-guided adrenalectomy is essential to identify patients with residual or recurrent PA, in order to initiate targeted medical therapy and to prevent cardiovascular and renal complications.

Nonstandard abbreviations and acronyms

A5

American-Australian-Asian Adrenal Alliance

APA

aldosterone-producing adenoma

ARR

aldosterone-to-renin ratio

AVS

adrenal vein sampling

BB

bilateral PA on AVS without and with cosyntropin

BL

AVS lateralization only after cosyntropin

CSI

Contralateral suppression index

CYP11B2

aldosterone synthase

DRC

direct renin concentration

FFPE

formalin fixed paraffin embedded

IHC

immunohistochemistry

IQR

interquartile range

LB

AVS lateralization only at baseline

LI

lateralization index

LL

AVS lateralization before and after cosyntropin

NGS

next generation sequencing

OR

odds ratio

PA

Primary aldosteronism

PAC

plasma aldosterone concentration

PASO

primary aldosteronism surgical outcomes

PRA

plasma renin activity

Data Sharing

We will consider sharing deidentified, individual participant-level data that underlie the results reported in this Article on receipt of a request detailing the study hypothesis and statistical analysis plan. All requests should be sent to the corresponding author. The corresponding author and lead investigators of this study will discuss all requests and make decisions about whether data sharing is appropriate based on the scientific rigor of the proposal. All applicants will be asked to sign a data access agreement.