ACTH-dependent Cushing's syndrome (CS) is a diagnostic challenge. Clinical variability and difficult interpretation of diagnostic tests delay diagnosis and increase morbidity.

To describe clinical, biochemical and radiological differences in patients with ACTH-dependent CS, with the aim of determining the variables that may help to non-invasively differentiate between Cushing's disease (CD) and ectopic CS (ECS).

A retrospective study included patients with ACTH-dependent CS followed at the Ramón y Cajal University Hospital between 2018 and 2023.

Twenty-eight patients with CD and 9 with ECS were included. Urinary free cortisol (UFC) levels presented an area under the ROC curve (AUC) of 0.812 (95% CI, 0.607–1.000) to differentiate between the two entities. UFC > 4.2 times the upper limit of normal showed 62.5% sensitivity and 90.9% specificity for diagnosis of ECS. The differential diagnostic accuracy of potassium level between ECS and CD was 82% (95% CI, 0.598–1.000). The combination of UFC and potassium values increased diagnostic accuracy to 85% (95% CI, 0.586–1.000). 55.6% of patients with ECS presented metabolic alkalosis, compared to 2.8% of those with CD ( P = 0.001). Pituitary adenoma size presented an AUC of 0.844 (95% CI, 0.59–1.000) for diagnosis of CD. Size ≥ 4.5 mm presented 93.8% sensitivity and 50% specificity for the diagnosis of CD.

UFC, hypokalemia, metabolic alkalosis and pituitary adenoma size are the non-invasive parameters most accurately differentiating between CD and ECS.