A 49-year-old woman with no significant past medical or surgical history other than sporadic migraines presented to the emergency room at Jackson Memorial Hospital complaining of a 2-week episode of abdominal distention and flank pain. Initial examination revealed an otherwise normal female with vital signs within normal limits and discomfort in the right flank, suspicious for a kidney stone. As part of her initial workup, the patient had an abdominal CT scan that revealed a 15 cm right adrenal mass (Figure 1(a)). No stones or signs of hydronephrosis or pyelonephritis were identified. Laboratory workup, including serum determination of cortisol (5.7 mcg/dL, normal range: 4.3-22.4 mcg/dL at 8 am), aldosterone (<4.0 ng/dL, reference: <=21 ng/dL), and adrenocorticotropic hormone (12 pg/mL, reference: <47 pg/mL), was unremarkable. The patient underwent surgical excision of the mass. The resected specimen consisted of a well-encapsulated oval mass with a bright golden-yellow parenchyma. The right adrenal gland was found adjacent to the mass (Figure 1(b)). Microscopic examination of the tumor after formalin fixation demonstrated a neoplasm composed of large oncocytic cells (Figure 1(c)) with focal areas of nuclear pleomorphism (Figure 1(c), insert). The presence of increased intracytoplasmic mitochondria was confirmed by electron microscopy (Figure 1(d)). Mitotic figures were not observed. According to the proposed classification by Bisceglia et al., the tumor size and the absence of mitoses, necrosis, capsular, and sinusoidal invasion indicate that this AON could harbor borderline malignant potential. The patient's postsurgical course was unremarkable and no further treatment was required. Currently, four years after surgery, the patient is alive, tumor-free, and in her normal state of health.
In order to assess the expression of TSPO we obtained additional unstained slides from formalin-fixed paraffin-embedded (FFPE) tissue including a representative section of the tumor with adjacent normal adrenal gland (internal control) of the patient. Two slides were deparaffinized after incubation at room temperature (RT, 24 C) in xylene (twice for 10 minutes). The deparaffinized tissue sections were then rehydrated with a graded series of ethanol (100%, 100%, 70%, 70%, and 50%) and incubated in phosphate buffered saline (PBS) for 15 minutes at RT. After incubation, slides were stained for TSPO by immunofluorescence as previously described. Primary antibody to detect TSPO (FL-169, Santa Cruz Biotechnology, Inc., Dallas, Texas, cat# 20120) was used at 1 : 75 dilution, according to the manufacturer instructions. Fluorescent HRP-conjugated secondary antibody (Alexa Flour-488 goat anti-rabbit IgG (H+L)) was used at 1 : 200 dilutions. The slides were reviewed with a Zeiss LSM510/UV Axiovert 200 M confocal microscope (Carl Zeiss, Peabody, MA, USA). Multiple images captured from tumor cells and normal adrenal gland showed strong immunofluorescence positivity in the normal adjacent adrenal gland (Figure 2(a)) and significant loss of TSPO expression in the tumor cells (Figure 2(b)).
To more precisely evaluate the loss of nuclear and cytoplasmic TSPO expression in tumor cells and confirm the immunofluorescence findings, we dissected mapped tumor and normal adrenal tissue from FFPE unstained slides and performed immunoblots. Briefly, FFPE tissue sections were deparaffinized by incubation at RT in xylene (twice for 10 minutes). The deparaffinized tissue sections were then rehydrated with a graded series of ethanol (100%, 100%, 70%, 70%, and 50%) and incubated at RT in PBS for 15 minutes. After incubation, tumor and normal adrenal tissue were dissected off the slides and placed in two separate plastic tubes. The tubes were pelleted at 16,000 xg for 5 minutes, and the incubation/centrifugation steps were repeated twice. Tissue samples were briefly air-dried in a fume hood. The cell pellet was resuspended in 200 mul cold buffer A, consisting of 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 muM dithiothreitol (DTT), and a complete protease inhibitor cocktail (Roche, Mannheim, Germany). The pellet was then incubated on ice for 15 minutes to allow cells to swell, after which 15 mul of 10% NP-40 was added, and the sample was vortexed thoroughly for 40 seconds and centrifuged at 3,000 rpm for 3 minutes at 4 C. The resulting supernatant was used for cytosolic TSPO measurement (equal amount of protein, 12.4 mug was loaded on an SDS-polyacrylamide gel and Western blot analysis with TSPO antibody was performed as described previously) and the pellet (nuclear fraction) was resuspended in 30 mul cold buffer B consisting of 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 muM DTT, and protease inhibitors. The pellet was then incubated on ice and vortexed for 15 seconds every 2 min for up to 15 min. The nuclear extract was then centrifuged at 13,000 rpm for 5 minutes at 4 C. Equal amounts of protein (21.6 mug) from the supernatant (containing the nuclear extract) were loaded and Western blot analysis with TSPO antibody was performed as described above. The quality of the nuclear extract was analyzed by propidium iodide staining, which indicated a purity of 92-96%. Primary TSPO antibody (Santa Cruz Biotechnology, Inc., Dallas, Texas, cat# 20120) was used at 1 : 1000 dilution. Beta actin (ACTBD11B7, sc-81178, Santa Cruz Biotechnology, Dallas, TX, USA) and lamin a/c (Cell Signaling Technology, Beverly, MA, USA) antibodies were used at 1 : 5000 and 1 : 750, respectively. Anti-rabbit and anti-mouse secondary antibodies (Vector Laboratories, Burlingame, CA, USA) were used at 1 : 3000 dilution. Optical density of the bands was determined with the Chemi-Imager (Alpha Innotech, San Leandro, CA, USA) digital imaging system and the results were quantified with the Sigma Scan Pro (Jandell Scientific, San Jose, CA, USA) program as a proportion of the signal of housekeeping protein bands (lamin a/c and beta-actin, nuclear and cytosolic markers, resp.). The experiment was performed using 4 different tissue sections from the same sample and the mean intensity of Western blot bands was subjected to Tukey's multiple comparison test. Statistical significance was set at p value = 0.05. As illustrated in Figures 2(a) and 2(b) (cytosolic fraction) and Figures 2(c) and 2(d) (nuclear fraction), the representative semiquantitative immunoblots from two different tumor sections show a significant decrease in TSPO expression in the tumor sample of 72.4 and 72.8% decrease in the cytosol and 77.1 and 76.8% decrease in the nuclear fraction as compared to respective controls (p = 0.03).