Extended Data Fig. 5: Sub-types of APA, phenotype-genotype correlations and immunohistochemistry (IHC).

a. Frequency of APAs harbouring each of the known somatic gene mutations. [i] Each genotype was sub-divided to show variation in ethnicity, demonstrating significant differences between genotypes. n = 60. Fisher’s Exact test p = 0.0312. Genotyping by sex is shown in [ii] male, n = 39 and [iii] female, n = 21 patients. A correlation between ethnicity and genotype was seen in female (p = 0.0014) but not male patients (p = 0.3261). b. CYP11B1 and CYP11B2 mRNA expression by APA genotype. mRNA expression of APAs is the normalised read count on RNA sequencing. The bar represents mean and error bars show +/− 1 standard deviation from mean. For CYP11B2 expression, KCNJ5 v other genotypes, Kruskal-Wallis test, p = 2.95×10^-7; For CYP11B1 expression, KCNJ5 v other genotypes, Kruskal-Wallis test, p = 2.0×10^-5. Total with genotype and gene expression, n = 51. Numbers of each genotype are smaller than in Extended Data Fig. 5a, because not all adrenals had an RNAlater sample collected at the time of surgery for RNA sequencing. c. Intensity of CYP11B1 and CYP11B2 expression of APAs on IHC staining was quantified using the H-score system⁶⁹. The average H-score for each genotype showed differences in CYP11B1 expression between KCNJ5-mutant and other APAs. ANOVA p = 0.0002; KCNJ5 v CACNA1D p = 0.0009, KCNJ5 v ATP1A1 p = 0.0128, KCNJ5 v ATP2B3 p = 0.0022. No differences in CYP11B2 expression on IHC was detected between genotypes. ANOVA p = 0.937. Paired comparisons were made using Tukey’s honestly significant difference test. Note small sample size within each group in above analysis (n = 3-9), although these did pass the Shapiro-Wilk test for normality. H-scoring only performed on APAs of known gene mutations from Cambridge University Hospital, total. n = 30. d. Two illustrative cases demonstrating selective [¹¹C]-metomidate uptake in patients with two or more adrenal adenomas. [i] Patient 1 has two adenomas on their adrenal, shown in separate axial MTO images. The first nodule is [¹¹C]-metomidate avid while the second is ‘cold’. IHC staining of the two nodules show strong positive staining for CYP11B2 in nodule 1 while nodule 2 is negative for CYP11B2. IHC staining for CYP11B1 is positive in both nodules. [ii] Patient 2 had multiple adrenal nodules, only one of which (nodule 1) demonstrated high [¹¹C]-metomidate uptake on PET-CT (left hand panels). Top middle and top right hand panels show high power views of patient 2’s adrenal, with CYP11B2 and CYP11B1 IHC staining respectively. Bottom middle panel: RNA sequencing results showing increased mRNA expression for CYP11B1 (compared to AAG) in both nodules, and increase mRNA expression for CYP11B2 in nodule 1 only. Scale bar represents 5 mm. APA, aldosterone producing adenoma; AAG, adjacent adrenal gland; IHC, immunohistochemistry; NFA, non-functioning adenoma.