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Biochemical Assessment of Cushing’s Disease in Patients with Corticotroph Macroadenomas¹

Neuroendocrine Unit (L.K., M.S., A.K.) and Endocrine Division (J.S.B., J.R.T.), Department of Medicine, General Clinical Research Center (D.A.S.), Department of Neurosurgery (N.T.Z., B.S.), and the Neuropathology Department (E.T.H.W., D.W.H.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Laurence Katznelson, M.D., Neuroendocrine Unit, Bulfinch 457, Massachusetts General Hospital, 32 Fruit Street, Boston, Massachusetts 02114.

	Abstract

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The majority of cases of Cushing’s disease are due to an underlying pituitary corticotroph microadenoma (10 mm). Corticotroph macroadenomas (>10 mm) are a less common cause of Cushing’s disease, and little is known about specific clinical and biochemical findings in such patients. To define further the clinical characteristics of patients with corticotroph macroadenomas, we performed a retrospective review of Cushing’s disease due to macroadenomas seen at Massachusetts General Hospital between 1979 and 1995. Of 531 patients identified with a diagnostic code of Cushing’s syndrome, 20 were determined to have Cushing’s disease due to a macroadenoma based on radiographic evidence of pituitary adenoma greater than 10 mm and pathological confirmation of a pituitary adenoma. A comparison review of charts of 24 patients with Cushing’s disease due to corticotroph microadenomas identified on the basis of radiographic evidence of a normal pituitary gland or a pituitary adenoma 10 mm or less in diameter was also performed.

The mean ages of the patients (±SD) with macroadenomas and microadenomas were similar (39 ± 12 and 38 ± 14 yr, respectively). The baseline median 24-h urine free cortisol (UFC) excretion was 1341 nmol/day (range, 304–69,033 nmol/day) and 877 nmol/day (range, 293–2,558 nmol/day) for macroadenoma and microadenoma patients, respectively (P = 0.058). After the 48-h high dose dexamethasone suppression test, UFC decreased by 77 ± 19% (mean ± SD) and 91 ± 7% in macroadenoma and microadenoma subjects, respectively (P = 0.04). Fifty-six percent of macroadenoma patients and 92% of microadenoma patients had greater than 80% suppression of UFC after high dose dexamethasone administration (P = 0.03). The baseline median 24-h urinary 17-hydroxysteroid (17-OHCS) excretion was 52 µmol/day (range, 25–786 µmol/day) and 44 µmol/day (range, 17–86 µmol/day) for macroadenoma and microadenoma subjects, respectively (P = 0.09). After the standard high dose dexamethasone suppression test, 17-OHCS excretion decreased by 46 ± 33% and 72 ± 22% for macroadenoma and microadenoma subjects, respectively (P = 0.02). Fifty-three percent of patients with macroadenomas and 86% of patients with microadenomas had greater than 50% suppression of 17-OHCS after high dose dexamethasone administration (P = 0.02). Baseline plasma ACTH values were above the normal range in 83.3% of macroadenoma patients and in 45% of microadenoma subjects (P = 0.05).

Tumors were immunostained with the MIB-1 antibody for Ki-67 to investigate proliferation in the adenomas. There was a trend for a higher Ki-67 labeling index in corticotroph macroadenomas, and seven (44%) macroadenomas vs. three (18%) microadenomas had labeling indexes greater than 3%, but this was not statistically significant.

In summary, corticotroph macroadenomas are often associated with less glucocorticoid suppressibility than the more frequently occurring microadenomas. Therefore, the lack of suppression of UFC or 17-OHCS after the administration of high dose dexamethasone in a patient with Cushing’s disease does not necessarily imply the presence of ACTH-independent Cushing’s syndrome and is more commonly seen in patients with corticotroph macroadenomas than in those with microadenomas. Increased plasma ACTH concentrations are typical of patients with corticotroph macroadenomas and may be a more sensitive indicator of neoplastic corticotrophs than the UFC or 17-OHCS response to standard high dose dexamethasone testing.

	Introduction

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CUSHING’S disease is defined as hypercortisolism due to chronic overproduction of ACTH by a corticotroph adenoma. The majority of corticotroph adenomas are less than 10 mm in diameter and are described as microadenomas. The diagnostic hallmark of Cushing’s disease is the diminished suppressibility of ACTH and cortisol in response to exogenous glucocorticoids, such as dexamethasone (1, 2). In corticotroph adenomas, the set-point for ACTH suppressibility by glucocorticoids is altered, as demonstrated by the need for larger doses of exogenous glucocorticoids to reduce serum ACTH levels. Cortisol nonsuppressibility during administration of low dose dexamethasone (0.5 mg orally every 6 h for 48 h) but suppressibility during high dose dexamethasone (2.0 mg orally every 6 h for 48 h) is the key diagnostic finding in patients with Cushing’s disease in approximately 90% of cases (3). The altered set-point for glucocorticoid feedback on ACTH release in corticotroph adenomas is the basis for the diagnostic localization of the pathological lesion to the pituitary gland in patients with ACTH-dependent Cushing’s syndrome. This contrasts with the lack of glucocorticoid suppressibility typically found in patients with ACTH-independent hypercortisolism.

Less commonly, Cushing’s disease has been reported in the setting of a pituitary corticotroph macroadenoma, defined as a tumor diameter greater than 10 mm (4, 5). These case reports suggest that corticotroph macroadenomas have clinical and biochemical manifestations different from those of microadenomas, including reduced ACTH and cortisol suppressibility after standard dexamethasone testing (6). There have been no comprehensive studies characterizing corticotroph macroadenomas. To define the clinical and biochemical characteristics associated with corticotroph macroadenomas, we analyzed retrospectively hospital charts of patients with Cushing’s disease from the Massachusetts General Hospital to determine 1) clinical characteristics of patients with pituitary macroadenomas compared with those with microadenomas, and 2) glucocorticoid responses to dexamethasone testing in patients with macroadenomas. To determine whether corticotroph macroadenomas exhibit different proliferative or histological characteristics compared with microadenomas, we performed pathological analysis on adenoma specimen from subjects identified by this chart review using a sensitive marker of cell proliferation.

	Materials and Methods

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We reviewed the records of 531 patients with an ICD-9 code for Cushing’s syndrome who were seen at Massachusetts General Hospital between 1979 and 1995. We then identified patients with Cushing’s disease due to a pituitary corticotroph macroadenoma based on the following inclusion criteria: 1) radiological evidence (by computed tomography or magnetic resonance imaging) of a pituitary tumor greater than 10 mm in at least one dimension, and 2) pathologic documentation of a pituitary adenoma. If the radiological report did not include tumor dimensions, and copies of the scans were not available for our interpretation, then tumors were included as macroadenomas only if there was clear evidence of significant suprasellar extension and/or cavernous sinus invasion. Patients were excluded if they had undergone previous pituitary radiation or adrenalectomy. To compare biochemical and pathological evaluations in patients with macroadenomas with those of a comparable number of patients with microadenomas, charts of patients identified above with Cushing’s disease who were seen in the Neuroendocrine Clinical Center at Massachusetts General Hospital were reviewed in alphabetical order. Inclusion criteria for these patients with corticotroph microadenomas included 1) radiological evidence of a pituitary tumor 10 mm or less in diameter or a normal scan, and 2) pathological evidence of a pituitary adenoma and/or biochemical cure after surgery (cure was defined by urinary free cortisol (UFC) below 55 nmol/day or fasting serum cortisol below 83 nmol/L while receiving less than 1.0 mg oral dexamethasone/day). Subjects with a history of alcohol abuse or who had received supraphysiological glucocorticoid therapy within 1 yr preceding biochemical testing were excluded. The percent suppression of each steroid after administration of high dose dexamethasone was calculated for each patient using 24-h urine steroid collections with the following formula: [(baseline UFC or 17-OHCS) - (UFC or 17-OHCS on day 2 of dexamethasone)/(baseline UFC or 17-OHCS)] x 100. The study was approved by the subcommittee on human studies at the Massachusetts General Hospital.

Ki-67 staining

Sections of formalin-fixed, routinely processed, and paraffin-embedded tumor tissue were used for MIB-1 immunostaining. Sections were microwaved, after deparaffinization and methanol/H₂O₂ blocking, in 10 mmol/L citrate buffer (pH 6.0) for approximately 10 min. The antigen retrieval procedure by microwave was performed as previously described (7). After a 30-min cooling and a thorough wash with distilled water, normal horse serum was used to block nonspecific reactions. Sections were then incubated with the monoclonal antibody MIB-1 (mouse IgG1, Immunotech, Westbrook, ME) at a dilution of 1:75 overnight at 4 C. The monoclonal antibody MIB-1 was raised against a human recombinant peptide corresponding to a 1002-bp Ki-67 complementary DNA fragment, and it recognized native Ki-67 antigen as well as the recombinant fragment of the Ki-67 molecule. Tissue sections were then incubated with a biotinylated horse antimouse secondary antibody and the avidin-biotin-peroxidase complex according to the standard protocol, reacted with 3,3'-diaminobenzidine-H₂O₂, counterstained with hematoxylin, dehydrated, cleared, and mounted.

Control materials consisted of sections of normal human tonsil that were included in each batch of Ki-67 staining.

Counts for Ki-67-positive tumor nuclei were taken at x400 using an ocular grid reticle. Depending on the size of the tumor section, a total of 1000–3000 tumor nuclei were counted from 4–7 microscope fields containing the highest density of stained tumor nuclei. The Ki-67 labeling index was expressed as a percentage of Ki-67-positive tumor nuclei per total tumor nuclei counted/specimen.

Statistical analysis

The Wilcoxon test was used to compare the distribution of baseline and percent suppressed UFC and 17-OHCS values and Ki-67 labeling indexes between patients with macroadenomas and microadenomas using two-sided P values. To allow for the comparison of biochemical tests for macroadenomas and microadenomas at multiple levels of dexamethasone suppression, distribution curves were constructed by plotting the sensitivity (percentage of patients who suppressed) against percent suppression cut-off values. Although baseline biochemical tests were performed using different assays, these values were pooled for analysis. Spearman correlation coefficients were used to test for associations between variables. Data are presented as the mean ± SD unless otherwise noted. Fisher’s exact test was used to compare gender distributions. Because of significant variability in ACTH assays during the study period, serum ACTH values were compared between the groups by Fisher’s exact test based on whether the values were elevated or normal using individual laboratory reference ranges. Differences in ages between the groups were compared with Student’s t tests.

	Results

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Clinical characteristics at diagnosis

Of the 531 patients identified with an ICD-9 code diagnosis of Cushing’s syndrome, 261 had Cushing’s disease. Twenty patients met the study inclusion criteria for corticotroph macroadenomas and were analyzed. These subjects had undergone biochemical assessment and surgery between the years 1985 and 1994. Control data were obtained from 24 patients with Cushing’s disease due to a corticotroph microadenoma identified at the Neuroendocrine Clinical Center. These subjects underwent biochemical assessment and surgery between the years 1987 and 1997. The ages of patients with macroadenomas vs. microadenomas were 39 ± 12 vs. 38 ± 14 yr, respectively (P = NS), and there were no differences in gender distribution (75% vs. 83% women, respectively). Visual field deficits were present in 6 (33%) of the 18 subjects with macroadenomas who had available data, but in none of the patients with microadenomas.

Baseline UFC data were available in 18 of the subjects with macroadenomas and all of the subjects with microadenomas. In the macroadenoma group, the median UFC concentration was 1341 nmol/day and ranged from 304–69,033 nmol/day. In the microadenoma group, the median UFC level was 877 nmol/day and ranged from 293–2,558 nmol/day (P = 0.058 compared to macroadenoma group).

Baseline 17-OHCS excretion data were available in 18 of the macroadenoma and 16 of the microadenoma subjects. In the macroadenoma group, the median 17-OHCS excretion was 52 µmol/day and ranged from 25–786 µmol/day. In the microadenoma group, the median urinary 17-OHCS excretion was 44 µmol/day and ranged from 17 to 86 µmol/day (P = 0.09 compared to macroadenoma subjects).

Preoperative serum ACTH concentrations were obtained in 18 macroadenoma and 22 microadenoma patients. Fourteen (78%) of the 18 macroadenoma subjects and 10 (46%) of the 22 microadenoma patients had serum ACTH levels above the normal range (P = 0.05).

Response to high dose dexamethasone

UFC measurements after the administration of high dose dexamethasone were available in 16 macroadenoma and all microadenoma patients, and 17-OHCS excretion measurements were determined in 15 macroadenoma and 14 microadenoma patients. The percent suppressions of UFC and 17-OHCS excretion for both groups are shown in Fig. 1. Overall, UFC decreased by 79 ± 19% in macroadenoma subjects and by 91 ± 7% in microadenoma patients (P = 0.04). 17-OHCS excretion decreased by 46 ± 33% in macroadenoma subjects and by 72 ± 22% in microadenoma patients (P = 0.02). The sensitivity for suppression of each biochemical marker after the high dose dexamethasone test in both groups of patients was assessed by constructing distribution curves, as shown in Fig. 2. A more profound degree of suppression of both UFC and 17-OHCS excretion was found in microadenoma compared to macroadenoma patients at all percent cut-off points. Nine (56%) macroadenoma subjects vs. 22 (92%) microadenoma patients demonstrated at least 80% suppression of UFC values after administration of the high dose dexamethasone test. In addition, 8 (53%) macroadenoma subjects and 12 (86%) microadenoma patients demonstrated at least 50% suppression of 17-OHCS excretion during this test. Baseline UFC and 17-OHCS excretion did not correlate with the magnitude of the percent suppression.

View larger version (20K): [in this window] [in a new window]   Figure 1. Percent suppression of UFC and 17-OHCS secretion after a standard high dose dexamethasone suppression (2.0 mg, orally, every 6 h) in patients with pathologically confirmed corticotroph macroadenomas and microadenomas.

View larger version (18K): [in this window] [in a new window]   Figure 2. Distribution curves for comparison of glucocorticoid suppressibility in patients with corticotroph macroadenomas and microadenomas. The sensitivity (percentage of patients who suppressed at each percent suppression cut-off) is plotted against percent suppression cut-off values. Solid line, Macroadenomas; dashed line, microadenomas.

Staining for Ki-67 was performed on 16 macroadenomas and 17 microadenomas. Ki-67 labeling indexes for corticotroph microadenomas and macroadenomas are shown in Fig. 3. There was a trend toward a higher labeling index in corticotroph macroadenomas (P = NS). Seven (44%) macroadenomas vs. 3 (18%) microadenomas had labeling indexes greater than 3%. However, the groups were not significantly different. Three (19%) macroadenomas and 2 (12%) microadenomas did not stain for Ki-67. The Ki-67 labeling index did not correlate with baseline steroid excretion or suppressibility of UFC or 17-OHCS in response to dexamethasone.

View larger version (19K): [in this window] [in a new window]   Figure 3. Ki-67 labeling indexes for corticotroph macroadenomas and microadenomas. The Ki-67 labeling index is expressed as a percentage of Ki-67-positive tumor nuclei per total tumor nuclei counted/specimen.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our series delineates further the prevalence and biochemical characteristics associated with corticotroph macroadenomas in Cushing’s disease. In previous reports, the prevalence of large pituitary corticotroph tumors has been documented in up to 10% of cases, although these studies were often performed with less sensitive radiological techniques (8). In a review by Aron et al. (6) in 1982 of 47 patients with Cushing’s disease, 8 (17%) subjects had tumors with extrasellar extension, suggesting the presence of macroadenomas. In a more recent series of 56 surgically resected corticotroph tumors, 3 macroadenomas were detected (4). In our study, approximately 9% of patients with Cushing’s syndrome had Cushing’s disease with a corticotroph macroadenoma. This number may actually underestimate the incidence of macroadenomas, because we excluded patients who did not have pathological confirmation of a corticotroph adenoma, had previously undergone radiation, or had inadequate radiological imaging necessary to confirm the presence of a macroadenoma. Such tumors may be associated with local mass effects, including compression of the optic chiasm and other cranial nerves (6). In our series, 33% of subjects with macroadenomas had evidence of visual field compromise. These data are similar to the incidence of optic chiasm compression seen with pituitary macroadenomas of different phenotypes (9, 10).

We found significantly less suppression of UFC and 17-OHCS after a standard high dose dexamethasone test in patients with macroadenomas compared to those with microadenomas. Gibson et al. (5) reported that plasma cortisol levels in two of four subjects with corticotroph macroadenomas did not change in response to high dose dexamethasone suppression testing. Aron et al. (6) described one patient with Cushing’s disease secondary to a macroadenoma whose UFC did not change in response to high dose dexamethasone testing. By employing a cut-off of 80% suppression of UFC after the high dose dexamethasone test for defining the presence of pituitary disease, Flack et al. (11) showed the sensitivity and specificity of the test to be 81% and 92%, respectively. Although 92% of patients in our study with microadenomas had 80% suppression during this test, similar to the findings of Flack et al. (11), only 56% of patients with macroadenomas suppressed to this level. Therefore, definitions of glucocorticoid suppressibility in response to the standard high dose dexamethasone test may need to be revised for these patients. It is possible that a subset of Cushing’s syndrome patients found to lack glucocorticoid suppressibility in the older literature may have had underlying macroadenomas.

Why do some patients with Cushing’s disease present with corticotroph macroadenomas and why are these tumors less suppressible after dexamethasone administration? The recent finding that corticotroph macroadenomas are monoclonal suggests that these tumors, like microadenomas, are a result of a sporadic mutation and subsequent clonal proliferation of neoplastic, corticotroph cells (3, 12, 13). A specific mutation(s) may occur in corticotroph cells, resulting in aggressive, monoclonal growth, leading to corticotroph macroadenomas with relative or absolute resistance to glucocorticoids. For example, mutations leading to tumor proliferation and glucocorticoid resistance may be important in the pathogenesis of corticotroph macroadenomas (14, 15, 16). Higher doses of dexamethasone may be required to overcome the new set-point for glucocorticoid suppression.

We investigated whether corticotroph macroadenomas have enhanced proliferative potential compared to microadenomas by assaying in situ the presence of Ki-67, a cell cycle antigen and a marker of cell proliferation (17). We found a trend toward a higher degree of proliferative activity of macroadenomas compared to microadenomas, which did not reach statistical significance. In previous studies, the degree of Ki-67 staining has been shown to correlate with aggression of various neoplasms, including gliomas and lymphomas (7, 18). In the normal pituitary gland, Ki-67 expression is either absent or restricted to a few isolated cells (19). In contrast, Ki-67 expression is detectable in both functioning and nonfunctioning pituitary adenomas, and the Ki-67 scores determined in corticotroph adenomas in our study are similar to those seen with pituitary tumors in other studies (19, 20, 21). Knosp et al. (21) showed that pituitary adenomas with dural invasion have higher proliferative activity based on Ki-67 staining than noninvasive adenomas. However, previous studies have not shown a marked effect of the proliferative index on pituitary tumor size (19, 20, 22). Additional investigations are necessary to understand the pathophysiology underlying the development of corticotroph macroadenomas.

Corticotroph macroadenomas may have biosynthetic defects in the processing of proopiomelanocortin to ACTH, resulting in lower plasma levels of bioactive ACTH in patients with these tumors relative to tumor size. Gibson et al. (5) recently demonstrated impaired processing of POMC to ACTH in six patients with corticotroph macroadenomas. Therefore, these tumors theoretically may grow to a larger size before the hypercortisolemia and clinical manifestations of Cushing’s syndrome are detected. The mechanisms underlying the altered glucocorticoid suppressibility in these tumors are unclear.

In summary, we have reviewed the charts of all patients with Cushing’s syndrome seen at our tertiary care referral center between 1979 and 1995 to determine the clinical and biochemical characteristics associated with corticotroph macroadenomas. In contrast to the more frequently occurring microadenomas, corticotroph macroadenomas are often associated with reduced glucocorticoid suppressibility. Therefore, the lack of suppression of UFC or 17-OHCS after the administration of high dose dexamethasone in a patient with a corticotroph macroadenoma can still be consistent with ACTH-dependent Cushing’s syndrome, particularly in the setting of a macroadenoma. Increased plasma ACTH concentrations are typical of patients with corticotroph macroadenomas and may be a more sensitive indicator of neoplastic corticotrophs than the UFC or 17-OHCS response to standard high dose dexamethasone testing.

	Footnotes

 
¹ This work was supported in part by a Clinical Associate Physician Award from the NIH and Grant M01-RR-01066.

Received January 5, 1998.

Revised February 18, 1998.

Accepted February 23, 1998.

	References

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