Adrenalectomy

A total of 82 fresh-frozen adrenal tissues, collected between October 2006 and October 2014, were used for the evaluation of livin, its isoforms α and β, CASP3, XIAP and DIABLO mRNA levels. In particular, 23 (NAG) deriving from the area surrounding the tumors (n = 20) or from adrenalectomies performed during surgery for renal carcinoma (n = 3) and 59 adrenocortical tumors (25 ACA and 34 ACC) have been investigated. Among these, 19 were paired samples of tumors and corresponding adjacent normal adrenal glands (13 ACA and 6 ACC). In a subgroup of 15 out of 19 paired samples with enough material (10 ACA and 5 ACC), livin protein expression was also investigated by WB analysis. Among the ACC group, 29 tissues were primary tumors, 1 local recurrence and 4 distant metastases. The last follow-up was January 2016. Patients undergone adrenalectomy for primary tumor presented a median follow-up of 31 months (range 3–189 months). Among these, 13 were died for the disease, 15 were still alive at the last follow-up and 1 was lost from the follow-up.
For the livin immunohistochemistry analysis, we investigated 314 paraffin-embedded tissue sections (including 192 ACC, 58 ACA, 20 NAG, 6 other normal tissues, 38 other cancers), comprising 171 standard full slides and 143 assembled in three tissue microarrays (TMA). TMA were assembled as previously reported [53 (link)] and only patients with at least 2 out of 5 evaluable cores in the TMA after the staining procedure were included in the final series. A total of 250 adrenocortical tumor samples were evaluated. These adrenocortical tumor tissues included 32 samples (17 ACA and 15 ACC) that had been investigated in a previous SNP array analysis [35 (link)] and which were used for the comparison between copy number alteration and protein expression. Among the ACC group, 147 tissues were primary tumors, 25 local recurrences and 20 distant metastases. The last follow-up was January 2016. Patient underwent to adrenalectomy for primary tumor presented a median follow-up of 37 months (range 1–224 months). Among these, 86 were died for the disease, 57 were still alive at the last follow-up and 4 were lost from the follow-up.
For the comparison between livin protein staining and mRNA levels, we evaluated a total of 31 tissue samples, including 10 ACA and 21 ACC. We also investigated other non-adrenal tissues, comprising 6 normal tissues (liver, ovary, uterus, stomach and 2 samples of tonsils) and 38 tissues from several cancers (melanoma, lymphoma, renal cell carcinoma, bladder cancer, colon cancer, hepatocellular cancer, pancreatic cancer, breast cancer, ovarian cancer, testicular cancer, prostatic cancer, bronchial cancer and non-small cell lung cancer) as controls.
Clinical parameters, such as sex, age at diagnosis, tumor size, hormone secretion pattern, pathological classification and, in case of ACC, tumor stage according to the European Network for the Study of Adrenal Tumors (ENSAT) classification [54 (link)], Weiss score, Ki67 proliferation index, presence and number of distant metastasis, clinical outcome were collected through the German ACC and the ENSAT Registry (www.ensat.org). Hormonal hypersecretion and malignancy of the tumors were defined according to established clinical, biochemical and pathological criteria [5 (link), 55 ]. Clinical parameters and tumor characteristics are summarized in Table 1.
The study was approved by the ethics committee of the University of Wuerzburg (No. 93/02 and 88/11) and written informed consent was obtained from all patients.

Our study used a validated algorithm to identify PA patients diagnosed in 1997–2010, and further enrolled PA patients aged ≥18 at the time of first medical record of PA (ICD code = 255.1). The administrative data on diagnosis and MRA prescription identified patients with primary aldosteronism in Taiwan had been validated17 (link). Figure 1 depicts our procedures for selecting study subjects. Our study only enrolled patients who ever used MRA (belonging to the ATC class C03D) in the one year prior to or the two years following the first ICD-9-CM coding of PA, because this additional condition could assure high values for both sensitivity and the positive predictive value according to our validated report17 (link). Two kinds of MRA drugs are listed in the guidelines for treating PA, and only one potassium sparing agent (spironolactone, ATC code = C03DA01) was available in Taiwan before 2011. We further separated PA patients into a group receiving adrenalectomy and another one receiving MRA treatment.

Bilateral adrenalectomy was performed in wild-type adult female mice. The adrenalectomized animals were supplied with 0.9% saline to maintain salt levels. One week after surgery, the AAV virus (AAV5-saCas9-Fshr) was stereotaxically injected into the pituitary to knock down the expression of Fshr. Mice were humanely euthanized if postsurgical complications progressed to a pre-defined humane endpoint at which the mice started to suffer.

This retrospective study was approved by the local ethics committee. Between February 2013 and February 2020, we retrospectively retrieved all patients that were operated on for adrenalectomy and received a definitive diagnosis upon histopathological examination of PCCs at the University of Padova. Recruited patients had to present the following criteria: (1) be evaluated at our third-level referral hospital and have undergone adrenal contrast-enhanced CT, including unenhanced and contrast-enhanced scans, with at least 5 mm slice thickness for unenhanced scan and 3 mm slice thickness for arterial and venous phases scans; (2) have a complete panel of hormonal secretion; (3) have received a histopathologically confirmed diagnosis of PCC after adrenalectomy and, if possible, the evaluation of the PASS [8 (link)]. The only exclusion criteria were (1) poor-quality CT images and (2) motion or breathing artifacts.