CXCR4-directed PET with ⁶⁸Ga-pentixafor versus adrenal vein sampling for the diagnosis of unilateral primary aldosteronism

Abstract

Objective

To explore the accuracy of unilateral primary aldosteronism (UPA) classification via adrenal vein sampling (AVS) and C-X-C chemoking receptor 4 (CXCR4)-directed positron emission tomography (PET) with ⁶⁸Ga-pentixafor (CXCR4-directed molecular imaging).

Methods

A retrospective cohort study was conducted with 89 patients who were diagnosed with UPA and who underwent unilateral adrenalectomy (ADX) at West China Hospital of Sichuan University from January 2021 to June 2023. For these patients, surgical decisions were made on the basis of either AVS (AVS group) or CXCR4-directed molecular imaging (CXCR4 group), and patients were regularly followed up for more than 6 months after surgery. Whether biochemical and clinical success was achieved 6 months after surgery was determined on the basis of the primary aldosteronism surgical outcomes (PASO) criterion. The complete success rates were compared between the AVS group and CXCR4 group. One-way analysis of variance was used to compare preoperative factors, postoperative biochemical success rates and clinical success rates between the two groups. Additionally, the postoperative outcomes of adrenal nodules of different sizes were compared.

Results

1. Among the 89 patients with UPA, 66 patients received ADX on the basis of AVS results, and 23 patients on the basis of CXCR4-directed molecular imaging results. The median age of the CXCR4 group [M (P25, P75): 45.00 years (39.00, 51.00)] was significantly lower than that of the AVS group [M (P25, P75): 49.00 years (40.75, 54.00)]. No significant differences in sex, history of hypertension, maximum blood pressure, antihypertensive drug defined daily dose (DDD), plasma aldosterone concentration (PAC), direct renin concentration (DRC), aldosterone-to-renin ratio (ARR), PAC after the captopril challenge test (CCT), PAC after the seated saline infusion test (SSIT), urea nitrogen, estimated glomerular filtration rate (eGFR), serum potassium level, diameter of the adrenal nodules or bilateral adrenal involvement were found. 2. There was no significant difference in the postoperative biochemical complete succcess rate (80.30 vs. 91.30%) or clinical cpmplete success rate (59.05 vs.65.21%) between the AVS group and the CXCR4 group. There were no significant differences in nitrogen, eGFR, serum potassium level, PAC, DRC, ARR, mean postoperative blood pressure or antihypertensive drug DDD after 6 months of follow-up. 3. For the identification of UPA patients with functional adrenal nodules ≥1 cm, CXCR4-directed molecular imaging have comparable diagnostic accuracies. 4. Three patients with adrenal micronodules achieved complete biochemical complete success after successful typing via CXCR4-directed molecular imaging, and unilateral aldosteronogenic micronodules were confirmed by immunohistochemistry (IHC) of CYP11B2 after surgery.

Conclusion

CXCR4-directed molecular imaging has high diagnostic value in diagnosing UPA. Patients with UPA diagnosed via CXCR4-directed molecular imaging achieved postoperative biochemical and clinical success, with outcomes that appear comparable to those of patients diagnosed on the basis of AVS. CXCR4-directed molecular imaging was more definitive for adrenal nodules larger than 1 cm.

Background

Primary aldosteronism (PA) is the most common form of secondary hypertension, and it is associated with more severe cardiovascular and kidney damage than essential hypertension [1]. Nearly 80% of UPA cases can be relieved or cured by unilateral ADX [2], but bilateral PA (BPA) patients generally require long-term treatment with minerocorticoid receptor antagonists (MRAs). A meta-analysis revealed that PA patients who underwent ADX had significantly lower rates of all-cause mortality and major cardiovascular events than those treated with MRAs and that the long-term survival benefits of ADX were significantly greater than the short-term perioperative risks [3]. To make more accurate treatment decisions, it is critical to classify UPA patients quickly and accurately. At present, AVS is recommended as the standard for typing and diagnosis worldwide [4–6]. However, AVS has several obvious limitations, such as invasive examination and radiation exposure, and the success rate and accuracy of the obtained results may be affected by multiple factors, such as differences in vascular development and cortisol fluctuations. CXCR4 is a G protein-coupled transmembrane receptor that is closely related to the expression of adrenal aldosterone synthetase (CYP11B2) [7]. Gallium-68 pentixafor (a synthetic ligand of CXCR4) specifically binds to the CXCR4 receptor on the membrane of aldosterone-producing adenoma (APA) cells, and PET imaging reveals high uptake, whereas normal adrenal cells and nonfunctional adenomas have low or no expression [7, 8]. This technological innovation provides new possibilities for typing of PA subtypes. To date, a small number of relevant studies worldwide have confirmed that CXCR4-directed molecular imaging can assist in the diagnosis of UPA [9], but diversify sample studies are still needed to confirm the value of this approach in the detection of protoaldehydes. In this study, we conducted a comparative analysis of UPA patients who had undergone unilateral ADX and retrospectively analysed the accuracy of CXCR4-directed molecular imaging in the typing and diagnosis of PA patients.

Subjects and methods

Study subjects

This was a retrospective cohort study. Patients with PA who had undergone unilateral ADX at West China Hospital of Sichuan University from January 2021 to June 2023 and met the study criteria were included. All the subjects signed informed consent forms. This study was approved by the Ethics Committee of West China Hospital of Sichuan University (Ethics number: 2019 Review No. 692).

Inclusion criteria

(1) All the subjects were screened and diagnosed in accordance with the screening and diagnostic procedures recommended by the European Society of Hypertension (ESH) consensus for PA [4] and previous studies at our center [10]: the captopril challenge test (CCT) or seated saline infusion test (SSIT) should be performed after screening ARR > 2.4 (ng/dl)/(mIU/L) (see Appendix 1 for the CCT and SSIT procedures). PA was diagnosed in patients with a PAC > 11 ng/dl in the CCT or a PAC > 10 ng/dl in the SSIT [4, 5, 11].

(2) Adrenal nodules with a diameter of more than 5 mm were identified via adrenal enhanced computed tomography (CT) examination.

(3) Successful AVS or CXCR4-directed molecular imaging was performed.

(4) Regular follow-up observation for at least 6 months after the procedure was performed.

Exclusion criteria

(1) Complicated cases with severe hepatic and renal insufficiency.

(2) Patients with a history of malignant tumours before enrolment.

(3) Patients with severe acute cardiovascular disease (acute myocardial infarction, stroke, or coronary artery surgery) within 3 months before enrolment.

(4) Pregnant or lactating women.

(5) Patients who had taken oral steroid hormones for a long time due to their condition;

(6) Patients with hypercortisolism: In patients with autonomous and excessive cortisol secretion, cortisol levels could not be suppressed to 50 nmol/L after a 1 mg dexamethasone overnight inhibition test.

Classification and diagnosis method

The decision to perform unilateral ADX was made after successful evaluation of the dominant side secretion via the following two methods.

AVS

The whole process was completed under the guidance of data subtraction angiography at West China Hospital of Sichuan University. Nonsynchronous blood collection was performed with continuous intravenous infusion of adrenocorticotropic hormone (ACTH). At the beginning of blood collection, 25 IU of ACTH diluent was injected intravenously, and blood samples were collected for the detection of cortisol and aldosterone behind the inferior vena cava and bilateral adrenal veins. A ratio of cortisol between the adrenal vein and inferior vena cava >3 indicated successful collection.

Result interpretation: The lateralization index (LI) is based on the aldosterone/cortisol (A/C) concentration in the successfully cannulated adrenal vein (AV), and an LI > 4 indicates that there is an aldosterone-producing dominant side [12].

$$\mathrm{LI}=\left[{\left(\mathrm{aldosterone}/\mathrm{cortisol}\right)}_{\mathrm{dominant}\mathrm{side}}/{\left(\mathrm{aldosterone}/\mathrm{cortisol}\right)}_{\mathrm{nondominant}\mathrm{side}}\right].$$

CXCR4-directed molecular imaging

All patients were tested in the Nuclear Medicine Department of West China Hospital of Sichuan University, which produced the Gallium-68 pentixafor reagent. Approximately 3–5.50 mci of the reagent was injected after drug production, and PET/CT or PET/MRI scans of the adrenal area were performed within 40–60 min after injection.

Results interpretation: The maximum standard uptake value (SUV_max) of adrenal lesions was >6.5, the ratio of the adrenal lesional SUV_max to the normal liver SUV_mean (LLR) was >2.5. Alternatively, the ratio of the adrenal lesional SUV_max to the contralateral adrenal tissue SUV_mean (LAR) was >2.4, which was interpreted as unilateral secretion [13].

Detection methods

PAC and DRC were analysed with a LIAISON automated chemiluminescence immunoassay kit purchased from Diasorin (Italy). The intrabatch variability of the PAC ranged from 2.1 to 4.2%, and the interbatch variability ranged from 5.8 to 10.8%. The intrabatch variability of the DRC ranged from 2.1 to 4.9%, and the interbatch variability ranged from 6.8 to 13.0%. The reference range of the PAC was as follows: recumbent position: 3–23.6 ng/dL, upright position: 3.0–35.3 ng/dL. The reference range of the DRC was as follows: recumbent position: 2.8–39.9 mIU/L, upright position: 4.4–46.1 mIU/L. Blood pressure, serum creatinine, eGFR, electrolytes, PAC, DRC, ARR, blood potassium, and antihypertensive drug use were recorded 6 months after surgery.

Pathological staining: Immunohistochemical analysis of paraffin masses from 3 different patients was performed, and immunohistochemical staining of adrenal sections, including HE, CYP11B1 and CYP11B2 staining, was performed using an automatic immunostaining system. Antibodies against CYP11B1 (MABS502) and CYP11B1 (MABS1251) were purchased from Millipore Corporation. Semiquantitative analysis was performed according to the HISTALDO (Histology of Primary Aldosteronism) consensus to diagnose unilateral APA, aldosterone-producing micronodules and aldosterone-producing diffuse hyperplasia [14].

Postoperative observation

Postoperative follow-up

General data (sex, age, height, body mass index (BMI), maximum blood pressure, and history of hypokalaemia), serum creatinine, eGFR, electrolytes, PAC, DRC, and ARR were followed up regularly after ADX.

Postoperative remission assessment

On the basis of an international cohort study on outcomes after adrenalectomy in unilateral primary aldosteronism surgical outcomes (PASO) [2], the criteria for clinical and biochemical benefits after surgery in our centre were formulated.

Clinical evaluation

① Clinical complete success: Postoperative blood pressure independent of hypotensive drugs could be maintained at less than 140/80 mmHg, and blood potassium could be maintained at more than 3.5 mmol/L without supplementation. ② Clinical partial success: Postoperative blood pressure was easier to control than before surgery, the dose of antihypertensive drugs was reduced, and blood potassium was maintained at more than 3.5 mmol/L without supplementation. ③ Clinical absent success: Postoperative antihypertensive drugs resulted in no significant change compared with preoperative drugs with or without hypokalaemia. Patients with complete or partial clinical success were considered to exhibit a clinical effective response after surgery.

Biochemical assessment

① Biochemical complete success: a. Postoperative orthostatic ARR ≤ 2.4 (ng/dl)/(mIU/L) with normal blood potassium. b. Patients with an ARR > 2.4 (ng/dl)/(mIU/L) with normal potassium and a PAC < 11 ng/dl after the CCT. ② Biochemical partial success: ARR > 2.4 (ng/dl)/(mIU/L) with normal blood potassium and PAC > 11 ng/dl after CCT, but the PAC decreased by 50% compared with the preoperative value. ③ Biochemical absent success: Patients who did not meet the above two conditions.

Surgical cure

Biochemical and clinical data revealed that both patients achieved complete success.

Statistical methods

SPSS 27.0 software was used for statistical analysis. ANOVA and Bonferroni analysis were used for comparisons between groups. The nonnormally distributed data are expressed as M (P25, P75), and an independent sample nonparametric test was used for comparisons between groups. The count data are expressed as relative numbers, and the χ² test with Fisher’s exact probability method was used for comparisons between groups. P < 0.05 was considered to indicate statistical significance.

Results

Subjects

From January 2021 to June 2023, 120 PA patients who signed informed consent forms were enrolled and underwent unilateral adrenal resection at the Department of Endocrinology and Metabolism of West China Hospital of Sichuan University. One patient had severe renal insufficiency (eGFR < 30), and 4 patients underwent surgery directly on the basis of adrenal CT; hormone examination results were excluded. There were 8 patients who also underwent AVS and ADX, but AVS was failed. Additionally, 18 patients with a follow-up time of less than 6 months were excluded. Finally, 66 patients in the AVS group and 23 patients in the CXCR4 group were included. The CXCR4 group included 7 patients with PET-CT data and 16 patients with PET-MRI data (the screening process is shown in Fig. 1).

Fig. 1.

Flow chart of screening of enrolled PA patients

Comparison of preoperative clinical features between the two groups

The preoperative general data of the AVS group and CXCR4 group were compared via ANOVA. The median age of the CXCR4 group (45.00 years) was significantly lower than that of the AVS group (49.00 years). In addition, sex, BMI, blood pressure, maximum blood pressure, history of hypokalaemia, antihypertensive drug DDD, urea nitrogen, serum creatinine, eGFR, PAC, DRC, ARR, PAC after CCT, PAC after SSIT, adrenal nodal size and bilateral adrenal involvement were comparable between the two groups (Table 1).

Table 1.

Comparison of preoperative clinical features, shown as M (P25, P75)

	By detection method				By the diameter of adrenal nodules
Variables	AVS group (N = 66)	CXCR4 group (N = 23)	F	P	Patients with nodules ≥1 cm (N = 74)	Patients with nodules 5–10 mm (N = 15)	F	P
Age (year)	49.00 (40.75, 54.00)	45.00 (39.00, 51.00)	5.422	0.022*	48.00 (40.00, 53.00)	51.00 (40.00,59.00)	0.326	0.570
Sex (male, n (%))	33 (50.00)	11 (47.82)	0.032	0.859	33 (44.59)	11 (78.57)	2.146	0.147
Body mass index (kg/m2)	23.88 (21.77, 27.39)	24.09 (19.57, 26.40)	0.314	0.577	23.95 (21.50, 27.06)	24.09 (22.21,24.09)	1.108	0.295
Systolic blood pressure (mmHg)a	154.00 (140.50, 168.00)	145.00 (137.00, 155.00)	2.803	0.098	150.00 (139.75,163.25)	155.00 (139.00, 164.00)	0.709	0.402
Diastolic blood pressure (mmHg)a	101.00 (92.00, 111.25)	96.00 (94.00, 105.00)	1.471	0.228	99.00(93.00,108.25)	102.00 (92.00, 114.00)	0.034	0.854
History of hypokalaemia, n (%)	52 (78.78)	16 (69.56)	0.794	0.375	55 (74.32)	13(86.67)	1.043	0.310
History of hypertension, n (%)	59 (89.39)	21 (91.30)	0.067	0.796	68 (91.89)	12(80.00)	1.043	0.310
Maximum systolic blood pressure (mmHg)	170.00 (150.00, 190.00)	160.00 (150.00, 170.00)	0.640	0.426	170.00 (150.00, 187.75)	170.00 (145.00,180.00)	0.022	0.882
Maximum diastolic blood pressure (mmHg)	100.00 (90.00, 110.00)	100.00 (93.75, 110.00)	0.405	0.526	100.00 (90.00, 110.00)	100.00 (97.00,110.00)	0.446	0.506
Antihypertensive drug DDDb	1.00 (0.80, 1.60)	1.00(1.00, 2.40)	1.952	0.166	1.00(1.00,1.65)	1.00 (1.00,2.20)	0.387	0.536
Urea nitrogen (mmol/L)	4.50 (3.70, 5.30)	5.20 (3.50, 6.30)	2.596	0.111	4.50(3.50,5.97)	5.00 (3.80,5.60)	0.148	0.701
Serum creatinine (umol/L)	71.50 (58.25, 84.75)	78.00 (55.00, 94.00)	2.022	0.159	72.00(56.00,87.75)	80.00 (64.00, 88.00)	0.030	0.864
eGFR [ml/(min.1.73m2)]	97.74 (82.61, 109.23)	99.00 (84.06, 106.80)	0.650	0.422	98.32(81.59,109.11)	87.83 (84.79, 109.24)	0.417	0.520
Serum potassium (mmol/L)	3.48 (3.15, 3.85)	3.50(3.10, 3.86)	0.544	0.463	3.47(3.14,3.81)	3.79 (3.46,4.10)	2.760	0.101
DRC (mIU/L)	1.59 (0.78, 3.71)	1.87 (1.03, 4.05)	0.434	0.512	1.48 (0.78, 3.57)	2.11 (1.23,5.05)	0.101	0.752
PAC (ng/dl)	27.20 (18.48, 39.55)	24.70 (15.70, 62.20)	0.414	0.521	27.90 (18.27, 53.45)	21.60 (14.90,27.70)	1.575	0.213
ARR [(ng/dl)/(mIU/L)]	15.65 (7.15, 46.75)	17.69 (5.86, 35.64)	0.564	0.455	20.22(7.44, 46.75)	7.52(6.60,15.63)	1.742	0.190
PAC after CCT (ng/dl)	30.70 (19.00, 43.50)	19.60 (11.80, 54.90)	0.091	0.764	31.25(17.93,48.83)	22.00(15.80,30.10)	1.429	0.236
PAC after SSIT (ng/dl)	21.60 (13.80, 32.30)	16.80 (10.75, 40.20)	0.454	0.503	22.00(13.60,34.90)	20.95 (12.55, 24.05)	1.024	0.315
Bilateral adrenal involvement(Yes, n (%))	16 (24.24)	6 (26.08)	0.536	0.466	19 (25.67)	3 (20.00)	0.212	0.647
Maximum diameter of adrenal nodules (mm)	14.00 (11.00, 16.00)	13.00 (11.00, 16.00)	0.001	0.982	14.50(12.00,17.00)	5.00(5.00,5.00)	/	/

Reference range of general parameters: urea nitrogen: 3.1–8.8 mmol/L; creatinine: 49–88 µmol/L; Serum potassium: 3.5–5.3 mmol/L; eGFR: 80–120 mL/(min.1.73m²)

ARR renin/aldosterone ratio, DDD defined average daily dose of antihypertensive drugs, DRC direct renin concentration, eGFR estimated glomerular filtration rate, PAC plasma aldosterone concentration, CCT captopril challenge test, SSIT seated saline infusion test

^*P < 0.05, there was a significant difference

^aBlood pressure values were obtained from office measurements

^bThe daily maintenance dose of common antihypertensive drugs is defined according to the requirements of (https://www.whocc.no/atc_ddd_index/). The DDD definitions for common antihypertensive drugs in our centre is included in Appendix 2-DDD values of common antihypertensive drugs

Remission of patients in both groups after ADX

According to the standard definition of PASO, 83.17% (74/89) of all patients achieved biochemical success, the biochemical complete success rate was 80.3% (53/66) in the AVS group and 91.3% (21/23) in the CXCR4 group, and the difference was not statistically significant (F = 1.464, P = 0.230) (Fig. 2). A total of 84.30% (75/89) of the patients were effectively treated by surgery, and the clinical effective rate was 81.82% (54/66) in the AVS group and 91.3% (21/23) in the CXCR4 group, with no statistically significant difference between the two groups (F = 0.729, P = 0.396). In the end, 60.67% (54/89) achieved surgical cure, and the cure rate of the AVS group was 59.10% (39/66), whereas that of the CXCR4 group was 65.22% (15/23), with no statistically significant difference between the two groups (F = 0.928, P = 0.338).

Fig. 2.

Biochemical and clinic success after ADX. Notes: AVS-clinic: Clinical evaluation of the AVS group; CXCR4-clinic: Clinical evaluation of the CXCR4 group; AVS-bio: Biochemical assessment of the AVS group; CXCR4-bio: Biochemical assessment of the CXCR4 group

ANOVA was used to compare the biochemical and clinical outcomes of the two groups at 6 months after surgery (Table 2). In terms of biochemical outcomes, there were no significant differences in DRC, PAC, ARR or serum potassium between the two groups after surgery. A comparison of the preoperative and postoperative changes (Δ = preoperatively to postoperatively) between the two groups revealed that the differences in the Δblood potassium values between the two groups were statistically significant and that the increase in blood potassium levels in the CXCR4 group after surgery was greater than that in the AVS group. In terms of clinical outcomes, there were no significant differences between the two groups in terms of postoperative blood pressure, changes in blood pressure, or the use of antihypertensive drugs. Compared with the biochemical success patients (74 cases), 15 patients did not achieve biochemical complete success after surgery. There was no statistical difference in the size of adrenal nodules (F = 2.507, P = 0.117), whether bilateral lesions (F = 1.246, P = 0.267), preoperative PAC level (F = 0.679, P = 0.412), preoperative DRC (F = 0.933, P = 0.337), preoperative ARR (F = 0.001, P = 0.970), preoperative hypertension (F = 0.092, P = 0.762), and preoperative hypokalemia (F = 0.231, P = 0.632).

Table 2.

Clinical and biochemical outcomes after unilateral ADX, shown as M (P25, P75)

		AVS group (N = 66)	CXCR4 group (N = 23)	F	P
Biochemical outcomes	DRC (mIU/L)	11.78(4.11,29.07)	15.63(7.83,25.18)	0.000	0.990
	PAC (ng/dl)	7.12(5.00,10.40)	6.33(4.24,10.10)	0.547	0.461
	ARR [(ng/dl)/(mIU/L)]	0.52(0.27,1.61)	0.57(0.16,1.19)	1.335	0.251
	K (mmol/L)	4.31(4.15,4.61)	4.33(4.19,4.57)	0.113	0.738
	ΔPAC	19.36(8.15,34.03)	16.03(8.4,52.40)	0.327	0.569
	ΔDRC	−9.35(−24.16,−2.27)	−12.08(−23.39,−6.33)	0.369	0.545
	ΔK	−0.69(−1.34,0.26)	−0.89(−2.12,−0.48)	5.289	0.024*
Clinical outcomes	Systolic blood pressure (mmHg)	120.00(120.00,135.00)	120.00(120.00,135.00)	0.038	0.847
	Diastolic blood pressure (mmHg)	80.00(80.00,90.00)	80.00(70.00,85.00)	0.936	0.336
	ΔSystolic blood pressure (mmHg)	27.50(13.00,42.25)	23.00 (17.00,35.00)	0.414	0.522
	ΔDiastolic blood pressure (mmHg)	20.05 (6.5,28.50)	19.00 (10.00,32.00)	0.024	0.877
	ΔDDDa	1.00(0.25,1.5)	1.00(0.40,2.00)	1.022	0.315

ARR renin/aldosterone ratio, DDD defined average daily dose of antihypertensive drugs, DRC direct renin concentration, PAC plasma aldosterone concentration

ΔPAC preoperative-postoperative change in PAC, ΔDRC preoperative-postoperative elevation in DRC, ΔARR preoperative- postoperative change in ARR, ΔK preoperative-postoperative change in potassium

*P < 0.05, there was a significant difference

^aThe daily maintenance dose of common antihypertensive drugs is defined according to the requirements of (https://www.whocc.no/atc_ddd_index/). The DDD definition of common antihypertensive drugs in our centre is presented in Appendix 2-DDD values of common antihypertensive drugs.

UPA diagnosis accuracy with adrenal nodules of different sizes

According to the results of the adrenal CT scan, 89 patients were divided into two subgroups: those with adrenal nodule diameters ≥1 cm and those with adrenal nodule diameters ranging from 5–10 mm (Table 1). A total of 74 patients (83.14%) had adrenal nodules ≥1 cm. No significant differences were found between the two subgroups in terms of age, sex, BMI, blood pressure, maximum blood pressure, history of hypokalaemia, antihypertensive drug DDD, urea nitrogen, serum creatinine, eGFR, PAC, DRC, ARR, PAC after CCT, PAC after SSIT, adrenal nodal size and bilateral adrenal involvement.

In the adrenal nodule ≥1 cm subgroup, 81.00% (60/74) achieved complete biochemical success after the operation. Among these patients, 18 out of 20 (90%) who underwent CXCR4-directed molecular imaging achieved biochemical comolete success, whereas 44 out of 54 (81.48%) who underwent AVS achieved biochemical complete success, and the difference between the two methods was not significant (F = 0.193, P = 0.662).

There were 15 patients (15.73%) whose adrenal nodule size was between 5 and 10 mm, and the postoperative biochemical success rate of those patients was 80.00% (14/15), biochemical partial success rate was13.33%(2/15). CXCR4-directed molecular imaging was performed for 3 patients with micronodules, all of whom experienced biochemical complete success. The CT images of three patients revealed micronodules on the enlarged adrenal glands, with CT values less than 10 HU. These micronodules exhibited high uptake of ⁶⁸Ga-Pentixafor, with SUV_max values of 16.44, 7.22 and 6.68, respectively, and the LAR values corresponding to the nodules were calculated to be 5.63, 5.73, and 5.38, respectively. Nonseodular adrenal tissue had low uptake of ⁶⁸Ga-Pentixafor, with an average SUV between 3 and 4. After postoperative pathological staining for CYP11B2, three patients were classified according to the WHO HISTALDO classification model of PA, among which two had aldosterone-producing micronodules (APMs) and one had aldosterone-producing diffuse hyperplasia (APDH) (Fig. 3).

Fig. 3.

Imaging and pathology images for a patient with adrenal micronodules. Note: These images are from a 24-year-old female who underwent ⁶⁸Ga-Pentixafor PET-MRI and right adrenalectomy. IHC-CYP11B2: Immunohistochemistry with antibodies against CYP11B2

Discussion

To improve the cure rate of PA, accurate classification is very important for clinical decision-making. At present, the main methods recommended for PA classification include adrenal CT and AVS, among which AVS is considered the most accurate classification method at present. However, AVS is an invasive interventional procedure with the risk of complications such as bleeding, thrombosis and renal infarction. The anatomical characteristics of the adrenal veins [15] and differences in operator skills lead to a high risk of catheterization failure. As a result, the success rate of catheterization varies greatly among centres (approximately 44 [16]–80% [17]). Moreover, the need for iodine-contrast tracers has hindered the use of this approach in patients with hyperthyroidism and renal insufficiency. In addition, nearly 8.6–12% of PA patients present with autonomic cosecretion of cortisol [18, 19], which may affect the interpretation of the results [20].

These factors greatly reduce the accuracy of classification and diagnosis, indicating that AVS is not a perfect tool, as was hope. Therefore, researchers have explored other typing and diagnosis methods in recent years, including ¹¹C-metomidate (¹¹C-MTO) PET [21, 22] and predictive scoring [23]. They reported that although these methods had some value in typing and diagnosis, the accuracy of these methods was still lower than that of AVS. Therefore, identifying a more accurate, noninvasive and simple typing method is important for clinical practice related to the diagnosis and treatment of primary aldosteronism.

CXCR4-directed molecular imaging, a new detection method, is a PET imaging technology that targets CXCR4 and has high specificity and selectivity for multiple myeloma, lymphoma and myelodysplastic tumours [24, 25]. It has also been shown to have broad application prospects in cardiology and inflammatory diseases [26, 27]. Recently, several studies have shown that it is also effective in the typing and diagnosis of PA patients [9, 28, 29]. A prospective study involving 100 patients with PA revealed a 90% coincidence rate between CXCR4-directed molecular imaging and AVS in the subtyping and diagnosis of unilateral aldosteronomas [9]. In two other studies, ⁶⁸Ga-pentixafor uptake imaging was consistent with CYP11B2-positive nodule immunohistochemistry of adrenal adenoma in PA patients [9]. These findings suggest that CXCR4-directed molecular imaging may be a potentially advantageous diagnostic method for PA.

To further explore the value of CXCR4-directed molecular imaging in the diagnosis of PA. We retrospectively included 89 PA patients who underwent unilateral adrenalectomy via AVS or CXCR4-directed molecular imaging. Except for age, there were no significant differences in clinical characteristics or biochemical indicators between the two groups at baseline, indicating that the two groups were comparable. After 6 months of follow-up, no significant differences were found between the AVS group and the CXCR4 group in terms of improvement in blood pressure, dosage of antihypertensive drugs, PAC, blood potassium, ARR or other parameters, including biochemical complete success rate (80.3 vs. 91.3%) and clinical complete success rate (81.82 vs. 91.3%), which suggested that CXCR4-directed molecular imaging is not inferior to AVS in terms of providing surgical guidance. Comparative analysis found that there was a statistical difference in the changes of blood potassium(ΔK) between the two groups. The postoperative blood potassium of patients in the AVS group may be higher than that before surgery, considering that some patients may have renal insufficiency or postoperative relative cortical insufficiency. This study confirmed that CXCR4-directed molecular imaging not only has high accuracy for UPA classification but is also simple, convenient, and noninvasive, allowing it to replace AVS as the preferred choice for PA classification and diagnosis.

UPA mainly comprises unilateral adenomas larger than 1 cm in diameter. At present, a few studies have shown that ⁶⁸Ga-pentixafor has a sensitivity of 92.40% and a specificity of 94.40% for APA with a diameter above 1 cm [30]. Similar to these studies, the accuracy of CXCR4-directed molecular imaging in our centre for the diagnosis of UPA ≥1 cm was 90% (18/20), which is comparable with that in the AVS group (44/54, 81.48%). Hence, our study further confirmed the high accuracy of CXCR4-directed molecular imaging for APA. In addition, UPA includes adrenal micronodules (APMs), which are characterized as nodules less than 5 mm in diameter and are even invisible on CT [31]. Patients with APM identified in one study also achieved greater biochemical and clinical success rates after surgical treatment [32], but no other studies of CXCR4-directed molecular imaging involved APM. Therefore, we further analysed 15 UPA patients with adrenal micronodules (5 to 10 mm in diameter), 3 of whom underwent CXCR4-directed molecular imaging. Owing to the small nodule diameter, the tracer agent could not effectively distinguish between normal adrenal tissue and the lesion, so the LAR could not be obtained. Therefore, we combined two semiquantitative methods, namely, the LLR and the lesional SUV_max, to determine unilateral dominance in patients who underwent surgery. Postoperative follow-up revealed that all three patients achieved biochemical complete success, confirming that CXCR4-directed molecular imaging is also suitable for the classification of micronodules. More prospective studies with larger samples are needed to confirm these findings.

Although a few studies have shown the accuracy of CXCR4-directed molecular imaging in the diagnosis of aldosterone-producing ademoma, only one immunohistochemical study has confirmed the correlation between the expression of CXCR4 and that of CYP11B2 [7], and the mechanism by which CXCR4 participates in the development of aldosterone-producing ademoma, remains unclear. At present, several case reports suggest that CXCR4-directed molecular imaging may also be valuable in the diagnosis of other adrenal cortical diseases. For example, one report showed that 6 adrenocorticol adenoma and 2 primary bilateral macronodular adrenal hyperplasia (PBMAH) patients had positive uptake of ⁶⁸Ga-pentixafor [33]. However, the uptake observed on CXCR4-directed molecular imaging in a pheochromocytoma patient was lower than that in normal adrenal glands [34], suggesting that CXCR4 may also be involved in the formation of adrenal cortical and aldosterone-producing adenoma but is not associated with the occurrence of medullary tumours. However, more clinical investigations and basic studies are needed to confirm the accuracy of this hypothesis. CXCR4-directed molecular imaging may also have a significant effect on adrenal lymphoma, whose most common type is diffuse large B-cell lymphoma (DLBCL), and the expression of CXCR4, a tumour stem cell marker, in the cytoplasmic and/or plasmic chamber of DLBCL tumour cells is 50% [35]. Therefore, this approach may provide a new diagnostic method for adrenal lymphoma. In summary, the diagnosis of adrenal cortical adenomas can be facilitated by CXCR4-directed molecular imaging, which is a simple and noninvasive imaging method.

In terms of safety, CXCR4-directed molecular imaging has been used for non-PA patients for many years. A literature review and analysis of approximately 76 studies involving 1300 patients with CXCR4-directed molecular imaging has been conducted, but no significant adverse reactions or safety issues have been reported thus far [28]. To date, all patients whose CXCR4-directed molecular imaging was completed by the research centre have had no obvious adverse events or safety risks. These findings suggest that CXCR4-directed molecular imaging technology is safe and reliable for clinical application.

The primary flaw of this study is that it is a retrospective study with a small sample size. Patients primarily choose between CXCR4-directed molecular imaging technology and AVS based on factors such as examination costs, reservation time, and risk of trauma. Patients with small adrenal nodules appear to be more likely to receive AVS, which may be due to the fact that AVS has been developed in our center for more than ten years, which may result in patient selection bias. We began by comparing the baseline data differences between the two groups, and the results of the statistical analysis and comparisons between the two groups were essentially consistent except for age. The significant age difference was thought to be due to a bias in that younger patients may be more receptive to new technology when providing informed consent. The secondary deficiencies were insufficient observation parameters of follow-up patients, failure to observe and analyze postoperative changes in cortisol and renal function of patients, and inadequate analysis of postoperative remission. In the future, our center needs to continue to collect relevant data, expand the sample size and conduct random controlled trial (RCT), and we look forward to having more data to update relevant studies and make up for these limitations.

In summary, our study revealed that CXCR4-directed molecular imaging had high accuracy in the diagnosis of aldosterone-producing adenoma, and the postoperative biochemical complete success rate and clinical complete success rate were comparable to those of AVS. CXCR4 receptor imaging was more definitive for adrenal nodules larger than 1 cm, but we also found that this technique is effective in identifying hyperfunctional micronodules of 5–10 mm, which can play an important role in the rapid and accurate classification of PA.

Appendix 1 Captopril challenge test and seated saline infusion test procedures

Captopril challenge test procedure: Fifty milligrams of captopril was administered orally at approximately 8:00 after 2 h in the upright position. Blood samples were collected before and 2 h after drug administration to detect PAC, direct rennin concentration (DRC), ARR, and serum potassium. During this period, the patient remained in a upright position with fasting.

Seated saline infusion test procedure: After the patient remained in a seated position for 2 h, 500 mL/h of normal saline was injected intravenously at approximately 8:00, and continuous infusion was performed for 4 h. Blood samples were collected before and after infusion to detect PAC, DRC, ARR, and serum potassium. During this period, the patient remained seated with fasting.

Appendix 2 References for the DDD values of the antihypertensive drugs used in this study

Principles: Defined daily dose (assumed average maintenance dose per day for a drug used for its main indication in adults [https://www.whocc.no/atc_ddd_index/]); the minimum maintenance dose for the specification is defined as 1. The DDD values of commonly used antihypertensive drugs are listed below

Irbesartan tablets (150 mg/d) = 1

Terazosin (5 mg/d) = 1

Prazosin tablets (6 mg/d) = 1

Sustained-release diltiazem capsules (90 mg/d) = 1

Nifedipine controlled release tablets (30 mg/d) = 1

Verapamil sustained-release tablets (0.24 g/d) = 1

Amlodipine besylate tablets (5 mg/d) = 1

Metoprolol tablets (47.5 mg/d) = 1

Levamlodipine benzenesulfonate (2.5 mg/d) = 1

Arolol hydrochloride tablets (20 mg/d) = 1

Valsartan (80 mg/d) = 1

Felodipine (5 mg/d) = 1

Losartan potassium (50 mg/d) = 1

Doxazosin (4 mg/d) = 1

Spironolactone (40 mg/d) = 1

Reserpine tablet (0.25 mg/d) = 1

Hydrochlorothiazide (25 mg/d) = 1

Dipyrazine (10 mg/d) = 1

Nifedipine sustained-release tablets (40 mg/d) = 1

Jastar (perindopril tert-butylamine tablets) (4 mg/d) = 1

Finelidone (20 mg/d) = 1

Furosemide tablets (40 mg/d) = 1

Irbesartan hydrochlorothiazide tablets (150/12.5 mg *1 d) (irbesartan 150 mg + hydrochlorothiazide 12.5 mg) = 1.5

Sacubitactril valsartan tablets (100 mg/d) (containing 51 mg valsartan) = 0.64

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by L.T., T.C. and Y.R. W.Z., S.S., H.T., Y.C. and R.T. participated substantially in patients care. All authors read and approved the final manuscript. All other authors have declare no conflicts of interest.

Funding

This article was supported by the National Key Research and Development Program of China (No.2021YFC2501601), and the Science and Technology Department of Sichuan Province project funding (NO.23ZDYF2116).

Data availability

Due to confidentiality agreements and ethical restrictions, the privacy of subjects is protected and detailed source data are not disclosed.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Informed consent

All procedures have been conducted as part of clinical routine care. Informed consent has been obtained from all subjects.

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of West China Hospital(Ethics number: 2019 Review No. 692).