Tissue specimens were obtained from twenty-two patients with adrenocortical tumors (Table 1). Most of them has been followed in our service since the initial diagnosis of adrenocortical neoplasm. A fraction of these patients were referred to us after undergoing adrenalectomy. Before surgery, informed consent was obtained from all patients or their parents. Ages varied from 2 to 46 years at the time of diagnosis, with 6 males and 16 females. Initial clinical presentation included Cushing's syndrome, adult female virilization, adult male feminization, hypertension, pseudoprecocious puberty, and only one patient with a nonfunctional tumor (Table 1). Based on their clinical behavior adrenal tumors were classified as nonmetastasizing (NM) or metastasizing/recurring (MR). Patients included in the NM group (n = 13) presented no disease recurrence or metastasis after a mean follow-up of 6.84 ± 1.44 years (Table 1). Conversely, except for patient 22, 9 patients in the MR group died within 1.56 ± 0.79 years since the initial diagnosis, after presenting tumor dissemination and/or recurrence (Table 1). Patient 22, whose clinical data have been previously reported 25, presented two independent tumors excised from both left and right adrenals in a two-year period. Subsequently solitary lung metastases were detected and surgically treated. He shows no signs of residual disease after a seven-year follow-up. A same pathologist reviewed histopathological data from all patients and, when sufficient data were available, calculated Weiss scores (Table 1).

Tumor samples were immediately frozen in liquid nitrogen at the time of surgery and stored until RNA extraction. Total RNA from tumor samples and from human adrenocortical NCI-H295A cells was extracted using a guanidinium-thiocyanate-based commercial kit (TRI-Reagent, Sigma, St. Louis, MO). A commercial preparation of adult human adrenal total RNA (Human Adrenal Gland Total RNA, Clontech, Palo Alto, CA) was used as normal control. Five micrograms of total RNA from each sample were treated with 0.5 U of DNAse I, (Promega Corp., Madison, WI) for 30 min at 37°C. DNAse-treated RNAs were reversely transcribed at 42°C for 75 min with 200 U of a RNase H^- MMLV reverse transcriptase (Superscript II, Life Technologies Inc. Gaithersburg, MD) in a final volume of 20 μl containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 1 mM of each dNTP, 100 mM dithiothreitol, 0.5 μg of oligo dT (Life Technologies Inc. Gaithersburg, MD), and 30 U of RNAse inhibitor (RNasin, Promega Corp., Madison, WI).

One tenth of RT products was amplified in a 50 μl reaction mix containing 20 pmol of each specific primer, 0.2 mM dNTP each, 50 mM KCl, 10 mM Tris-HCl (pH 9.3), 1.5 mM MgCl₂ and 5 U Taq Polymerase (Amersham-Pharmacia, Piscataway, NJ). Twenty microliters of the PCR product were loaded on a 1.5% agarose gel for electrophoresis and visualization with ethidium bromide. 194-bp GATA-4 and 285-bp GATA-6 cDNA fragments were amplified using oligonucleotides GATA4-F: 5'-TCCCTCTTCCCTCCTCAAAT-3', GATA4-R: 5'-TCAGCGTGTAAAGGCATCTG-3', GATA6 – F: 5'-GAGAAGATGGAAGGGAAGGG-3', GATA6-R: 5'-TGTGTACCAAATGGCCTCAA-3', designed according to human GATA-4 and GATA-6 cDNA sequences (GenBank accession n° D78260 and U66075, respectively). Cycling conditions for PCR were: 95°C, 3 min, followed by 40 cycles at 95°C, 30 sec, 56°C, 30 sec, 72°C, 30 sec, and a final extension cycle of 7 min at 72°C. Amplification of a 234 bp fragment from the human luteinizing hormone receptor (LHR) transcript was accomplished using oligonucleotides eLHR: 5'-AGACATTCCAAAGAGATTTC-3' and LHR2101R: 5'-GCAGTTACTGATGTAACAGTTAACAC-3'. Cycling conditions were essentially the same as those described for GATA cDNA amplification except for a 50°C annealing temperature. As we amplified only exonic sequences, samples were tested with and without reverse transcriptase in order to rule out genomic DNA contamination. Samples quantity and integrity were verified by amplification of a 259-bp cyclophilin cDNA fragment using oligonucleotides CYC-F: 5'-TTTCACAGAAATTATTCCAGGGTTT-3' e CYC-R: 5'-CAATATTCATGCCTTCTTTCACTT-3'. Direct sequencing was performed to confirm the identity of RT-PCR products.

Fifteen microliters of GATA-4, GATA-6, LHR and cyclophilin PCR products were mixed with 185 μL NaOH 0.4 M, EDTA 25 mM, heated 95°C for 2 min, and vacuum-transferred onto a nylon membrane (Hybond N+, Amersham, Piscataway, NJ). Probes were generated by radioactive PCR as follows: fragments corresponding to GATA-4, GATA-6, LHR, and cyclophilin were amplified by PCR using genomic DNA as template. The amplification conditions and primer sets were the same as those described in the previous section. These fragments were then cloned into the pCR2.1-TOPO T.A. cloning vector (TOPO TA Cloning kit, Invitrogen, Carlsbad, CA). The identity of the desired cloned inserts was verified by direct sequencing. A second PCR was performed in a 25 μl reaction mix containing 100 ng of plasmid (GATA-4-pCR2.1, GATA-6-pCR2.1, LHR-pCR2.1, and cyclophilin-pCR2.1), 20 pmol of M13F/R primers, 0.1 mM dNTP each, 50 mM KCl, 10 mM Tris-HCl (pH 9.3), 1.5 mM MgCl₂, 0.5 μl of [α-³²P] dCTP (3000 Ci/mmol), and 5 U Taq Polymerase (Amersham-Pharmacia, Piscataway, NJ). Cycling conditions were: 94°C, 3 min, followed by 30 cycles at 94°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec, and a final extension cycle of 7 min at 72°C. Probes were purified using Quiaquick PCR Purification kit (Quiaquick PCR Purification kit, Qiagen GmbH, Germany) after visualization in a 6% polyacrilamide gel. The membranes were pre-hybridized in 5 × SSPE, 5 × Denhardt's, 1% SDS, 5% dextran, 50% formamide, and ssDNA (200 μg/ml) for two hours at 42°C. The hybridization was carried out overnight at 42°C in the same solution after addition of the probes (100 ng). Membranes were rinsed once in 2 × SSC, 0.1% SDS for 15 min at room temperature and then washed three times in 0.1 × SSC, 0.1% SDS, first at room temperature for 15 min, and twice at 65°C for 30 and 15 min. Membranes were exposed overnight on a phosphorscreen and an autoradiography was obtained using a Storm 860 phosphorimager (Amersham Pharmacia Biotech, Piscataway, NJ). Densitometric analysis was carried out using ImageQuant 2.1 software (Amersham Pharmacia Biotech, Piscataway, NJ). Two normalizations were performed: 1 – densitometry data from tumor samples were normalized using the optical density from respective positive control, in order to correct any variation in transferring efficiency and probe activity. 2 – optical densities of GATA-4, GATA-6 and LHR were normalized for each tumor sample with corresponding cyclophilin densitometry data in order to obtain the relative expression levels. Intra and inter-assay coefficients of variation for GATA-4, GATA-6, and LHR are, respectively, 4.14% and 13.23%, 12.63% and 12.24%, 7.73% and 12.16%.

Relative expression of GATA-6, GATA-4 and LHR in non-mestasizing and metastasizing groups was compared using Student t test. The ability of empirically defined cut-off values of GATA-4, 6 and LHR transcripts to discriminate between these two groups was verified using Chi-square test.

The ability of both normal and tumoral human adrenal cortices and also a human adrenocortical cell line – NCI-H295A 3839 – to express GATA-6 and GATA-4 was assessed by PCR amplification of reversely transcribed total RNA. In order to enhance the detection sensitivity and to perform quantitative analysis of GATA-6 and GATA-4 accumulation, amplification products from a second RT-PCR were then vacuum-transferred to a membrane and subjected to dot-blot hybridization. Both GATA-6 and GATA-4 transcripts were detectable in normal adrenal and in NCI-H295A cells, with GATA-6 being more abundant than GATA-4 (Figure 1). Thus, both GATA transcripts are normally expressed in human adrenal and NCI-H295A cells. GATA-6 mRNA was detected in all 23 tumors, irrespective of their behavior (Figure 2). Sample 16 yielded no visible amplification product on agarose gel but was clearly positive for GATA-6 in dot-blot (Figure 2), illustrating the higher detection sensitivity of this method. Amplification product corresponding to GATA-4 cDNA was detected in 11 out of 13 NM tumors and in 9 of the 10 MR tumors (Figure 3). Although a faint band, with the expected size for GATA-4, was visible in the lane corresponding to sample 16 (Figure 3), no signal was detected on dot-blot. Given the higher sensitivity and specificity of dot-blot, this sample was considered negative and the band regarded as an artifact. Densitometric analysis of the autoradiogram provided quantitative information regarding accumulation of GATA transcripts (Table 2). Non-metastasizing tumors expressed more GATA-6 than metastasizing ones (respectively, 3.035 ± 0.111 and 2.568 ± 0.138, p = 0.032) while an opposite pattern was observed for GATA-4 (0.539 ± 0.143 and 1.776 ± 0.384, p = 0.003).

Gonadotropins have been shown to up-regulate GATA-4 in gonadal tumor cell lines 3032. In mouse Leydig cell lines, GATA-4 mRNA accumulation is stimulated by hCG 30. Since LH/hCG receptor has been detected in both normal human and tumoral mouse adrenal cortices 4041, we speculated that LHR expression might be correlated with the accumulation of GATA-4 in human adrenal cortex. For this reason, we investigated the expression pattern of LH receptor in our adrenocortical specimens. Amplification products of LHR transcript were observed in all tumors, and also in normal adrenal cortex and NCI cells (Figures 1 and 4). Samples displaying more abundant LHR transcripts were, in their majority, NM tumors. Nevertheless, no statistically significant difference in LHR mRNA accumulation was found (1.298 ± 0.24 and 0.757 ± 0.162, respectively for NM and MR groups, p = 0.209) in this group compared to the MR one. Samples expressing more abundant GATA-4 generally expressed smaller amounts of LHR (samples 15, 17, 19, 20, Figures 3, 4 and 6). Conversely, samples expressing smaller amounts of GATA-4 usually expressed LHR more intensely (samples 2, 5, 7, 8, 11, 13, Figures 3, 4 and 6).

Since the relative expression of GATA transcripts in the NM group is different from those in the MR group (Table 2) we examined whether a cut-off value for the expression of such mRNAs could be used as a predictor of tumor behavior. In our cohort, an empirically established expression level of GATA-4 above 1.77 is correlated with poor prognosis (p = 0.006) with a relative risk of 4.25 (95% CI: 1.508 – 4.25). Such a cut-off value, however, would lead to a false negative diagnosis of 4 out of 10 MR subjects (patients 14, 16, 21, and 22a). In contrast, no cut-off allowing a clear discrimination between favorable and unfavorable outcome could be determined for GATA-6.

We also verified whether LHR expression could serve as a predictor for tumor outcome. Although NM and MR groups showed no differences regarding the relative expression of LHR, expression below an empirical threshold of 0.53 was correlated with aggressive behavior (p = 0.002) with a relative risk of 5.333 (95% CI: 1.815 – 5.333). Combination of both GATA-4 and LHR did not improve any further the ability to discriminate tumoral behavior and clinical fate.