0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society
Vol. 85, No. 10 Printed in U.S.A.
Genetic and Histologic Studies of Somatomammotropic Pituitary Tumors in Patients with the “Complex of Spotty Skin Pigmentation, Myxomas, Endocrine Overactivity and Schwannomas” (Carney Complex)* SVETLANA D. PACK†, LAWRENCE S. KIRSCHNER, EVGENIA PAK, ZHENGPING ZHUANG,† J. AIDAN CARNEY, AND CONSTANTINE A. STRATAKIS Unit on Genetics and Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development (L.S.K., C.A.S.), Laboratory of Pathology, National Cancer Institute (S.D.P., E.P., Z.Z.), National Institutes of Health, Bethesda, Maryland 20892; and Emeritus Staff, Department of Laboratory Medicine and Pathology (J.A.C.), Mayo Clinic, Rochester, Minnesota 55905. ABSTRACT Carney complex (CNC) is a familial multiple neoplasia and lentiginosis syndrome with features overlapping those of McCuneAlbright syndrome (MAS) and other multiple endocrine neoplasia (MEN) syndromes, MEN type 1 (MEN 1), in particular. GH-producing pituitary tumors have been described in individual reports and in at least two large CNC patient series. It has been suggested that the evolution of acromegaly in CNC resembles that of the other endocrine manifestations of CNC in its chronic, often indolent, progressive nature. However, histologic and molecular evidence has not been presented in support of this hypothesis. In this investigation, the pituitary glands of eight patients with CNC and acromegaly [age, 22.9 ⫾ 11.6 yr (mean ⫾ SD)] were studied histologically. Tumor DNA was used for comparative genomic hybridization (CGH) (four tumors). All tumors stained for both GH and prolactin PRL (eight of eight), and some for other hormones, including ␣-subunit. Evidence for somatomammotroph hyperplasia was present in five of the eight patients in proximity to adenoma tissue; in the remaining three only adenoma
T
HE COMPLEX OF “spotty skin pigmentation, myxomas, endocrine overactivity, and schwannomas” or Carney complex (CNC) is a multiple endocrine neoplasia (MEN) and lentiginosis syndrome (1– 4) that is inherited in an autosomal dominant manner (5), and is genetically heterogeneous (6 – 8). Although GH and PRL secretion are frequently abnormal in affected patients (9, 10), clinical acromegaly or significant hyperprolactinemia and GH- or PRL-producing tumors, respectively, have been detected in less than one fifth of them (11, 12). The pattern of biochemical abnormalities of GH and PRL secretion without pituitary tumors (detectable Received February 28, 2000. Revision received June 16, 2000. Accepted June 29, 2000. Address correspondence and requests for reprints to: Constantine A. Stratakis, M.D., DSc, Unit on Genetics and Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N262, 10 Center Drive, MSC1862, Bethesda, Maryland 20892-1862. E-mail:
[email protected]. * Presented in part at the 80th Annual Meeting of The Endocrine Society, New Orleans, Louisiana, 1998 (P3-552). † Present address: National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland.
tissue was available for study. CGH showed multiple changes involving losses of chromosomal regions 6q, 7q, 11p, and 11q, and gains of 1pter-p32, 2q35-qter, 9q33-qter, 12q24-qter, 16, 17, 19p, 20p, 20q, 22p and 22q in the most aggressive tumor, an invasive macroadenoma; no chromosomal changes were seen in the microadenomas diagnosed prospectively (3 tumors). We conclude that, in at least some patients with CNC, the pituitary gland is characterized by somatotroph hyperplasia, which precedes GH-producing tumor formation, in a pathway similar to that suggested for MAS-related pituitary tumors. Three GH-producing microadenomas showed no genetic changes by CGH, whereas a macroadenoma in a patient, whose advanced acromegaly was not cured by surgery, showed extensive CGH changes. We speculate that these changes represent secondary and tertiary genetic “hits” involved in pituitary oncogenesis. The data (1) underline the need for early investigation for acromegaly in patients with CNC; (2) provide a molecular hypothesis for its clinical progression; and (3) suggest a model for MAS- and, perhaps, MEN 1-related GH-producing tumor formation. (J Clin Endocrinol Metab 85: 3860 –3865, 2000)
by common imaging modalities) and infrequent development of clinically significant acromegaly is reminiscent of McCune-Albright syndrome (MAS) (13–16). Genetic studies of tumors excised from patients with CNC have indicated that their abnormality may be in the molecular pathway that involves the stimulatory ␣-subunit of the guanine nucleotide-binding protein (Gs␣) (17, 18), the gene responsible for MAS (13). However, Gs␣ mutations per se were not present in a study of a series of CNC tumors (18). The pituitary gland is also regularly affected in MEN type 1 (MEN 1), a condition that shares a number of features with CNC and MAS, including acromegaly. However, a GH- or PRL-producing adenoma is present in almost all patients with MEN 1, including the very young, who have GH or PRL hypersecretion, respectively (19 –21). In the present study, we investigated the pituitary tissue in eight patients with CNC who had acromegaly and underwent transsphenoidal surgery (TSS). All patients who had extratumoral pituitary tissue excised (five of eight) had evidence of hyperplasia surrounding the adenomas; in all these cases, more than one tumor was present in the gland, identifying, thus, a multicentric process. Comparative
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genomic hybridization (CGH), a technique that examines large changes in tumor DNA (22–25), showed no alterations in three microadenomas (which were identified prospectively). In contrast, a macroadenoma, detected in a patient who presented with fully established acromegaly and required medical treatment after TSS, exhibited multiple DNA changes. These data suggest that, as in other endocrine glands affected in the disorder, adenohypophyseal cells in CNC likely undergo multicentric hyperplastic changes that may or may not lead to tumor formation, depending on the extent of genetic changes in pituitary tissue. Materials and Methods Patients The institutional review boards of the NICHD, NIH, and the Mayo Clinic approved the contact of families with CNC and the participation of patients and their relatives in the present study, after giving informed consent (protocol 95-CH-0059). All patients in the prospective study were seen by one of the authors (C.A.S.). Available histologic slides were rereviewed by one of the authors (J.A.C.). All patients were screened for the clinical manifestations of CNC according to the criteria established by Stratakis et al. (6). Seven patients were members of large CNC families, most of which have been described elsewhere (6); one patient did not have a family history of CNC (sporadic case) (Table 1). The diagnosis of acromegaly was based on clinical symptoms, results of oral glucose tolerance test and levels of insulin-like growth factor I (IGF-I), as described elsewhere (26, 27). The GH, IGF-I, and PRL levels at diagnosis are shown in Table 2. In all patients, GHRH levels were less than 22 ng/L (within the normal range). TSS was performed in patients who had a tumor visible in pituitary magnetic resonance imaging. Microadenoma was defined as a tumor with its greatest diameter less than 1 cm; a tumor with its greatest diameter equal to or greater than 1 cm was considered a macroadenoma. Cure was defined as postoperative GH serum levels below 1 ng/mL, and normalization of the IGF-I levels and GH responses to oral glucose tolerance test (data not shown).
Histological analysis and DNA extraction Tissue for genetic analysis was obtained at the time of surgery, frozen at ⫺70 C, and stored for later use. For light microscopy, formalin-fixed, paraffin-embedded sections were stained with hematoxylin and eosin, periodic acid-Schiff, and the Gordon-Sweet silver reticulin stain. For immunocytohistochemistry, the avidin-biotin-peroxidase complex technique was used in conjunction with antibodies against GH, PRL, ACTH, TSH, LH, FSH, and ␣-subunit. Sections were stained, and the location of the staining of the various antisera compared in consecutive sections. DNA was extracted from frozen tissue in a 0.7-mL solution of 50 mm Tris (pH 8.0), 100 mm EDTA, 100 mm NaCl, 1% SDS, and 0.5 mg/mL proteinase K (25). Samples were then extracted ⫻4 in phenol/chloroform, precipitated with ethanol, and resuspended in 1⫻ Tris-EDTA.
TABLE 1. Identification, age, and clinical manifestations of patients with CNC and acromegaly Patient
Age
Gender
Family
1 2 3 4 5 6 7 8
19 38 44 42 18 21 38 19
M F F M M F M F
CAR01 CAR110 CAR16 CAR102 CAR07 CAR07 CAR20 Sporadic case
Other manifestations
L, L, L, L, L L, L, L,
P P, Mx, A, T P, Mx, T P, Mx, T P, T P, Mx P, Mx
M, Male; F, female; L, lentigines; Mx, myxoma; P, primary pigmented nodular adrenocortical disease; T, thyroid nodules or thyroid cancer.
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CGH CGH was performed as described previously (22, 23). Control DNA was prepared from peripheral blood lymphocytes of a cytogenetically normal male. Nick translation was performed to label tumor DNA with bio-16-dUTP and control DNA with digoxigenin-11-dUTP (Boehringer Mannheim, Mannheim, Germany). Five hundred nanograms of each of the labeled genomes were hybridized in the presence of excess Cot-1 DNA (50 g) (Life Technologies, Gaithersburg, MD) to metaphase chromosomes prepared from a karyotypically normal female donor (see Fig. 2). The biotin-labeled tumor genome was visualized with avidin conjugated to fluorescein isothiocyanate (Vector Laboratories, Inc., Burlingame, CA), and the digoxigenin-labeled control DNA was detected with antidigoxigenin rhodamine (Boehringer-Mannheim). Chromosomes were counterstained with 4⬘,6-diamidino-2-phenylindole and embedded in antifading agent to reduce photobleaching. Gray scale images of the fluorescein isothiocyanate-labeled tumor DNA, the tetra-methylrhodamine isothiocyanate-labeled control DNA, and the 4⬘,6-diamidino2-phenylindole counterstain from at least eight metaphases from each hybridization were acquired with a cooled charge-coupled device camera connected to a Zeiss microscope equipped with fluorochromespecific optical filters. Quantitative evaluation of hybridization was obtained, and average ratio profiles were computed as the mean value of at least eight ratio images, to identify chromosomal copy number changes (24, 25).
Results Clinical information and pituitary gland histology
GH-producing tumors were identified in all eight patients with clinically diagnosed acromegaly. All tumors stained for PRL and occasionally for other hormones (see below). Three of four patients who had acromegaly as the primary manifestation of CNC (cases 1, 5, and 6) had macroadenomas. Microadenomas were detected in all operated patients in the CNC prospective study (cases, 2, 3, 4, and 8). One of these patients (case 2) had been administered octreotide for approximately 6 months because of high GH levels; octreotide was discontinued 3 yr before TSS. Acromegaly was cured by TSS in all patients with microadenomas. Only one (of three) patient with macroadenoma was cured surgically (case 5). Multiple macroscopic and microscopic tumors were seen in the pituitary gland of five patients (cases 1 and 4 –7), including one with a microadenoma (case 7); in these patients, extratumoral pituitary parenchyma showed evidence of GH- and PRL-producing cell hyperplasia (see below). In three patients whose microadenomas (cases 2, 3, and 8) were excised completely, extratumoral parenchyma was not available for study. Adenohypophyseal hyperplasia, characterized by poorly delineated zones with increased cellularity and an expanded, somewhat irregular reticulin pattern was seen in five cases (Fig. 1.1). A zone of probable transition from hyperplasia to adenoma, characterized by the gradual disappearance of the reticulin pattern and increasing cellularity, was also documented in these cases (examples are shown in Fig. 1.2 and 1.4). Both hyperplastic areas and adenoma tissue stained for GH and PRL. PRL staining was less intense and more limited than GH staining (Fig. 1, 5, 6, 8, and 9), although in all cases it was the same cellular population that demonstrated immunoreactivity for both GH and PRL at consecutive sections. Staining for ␣-subunit was also present in three of the five tumors (Table 2 and Fig. 1.10) in the same pattern as that of PRL. Occasional cells stained for TSH (-subunit); LH was
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TABLE 2. Clinical course and biochemical, histologic, and genetic analysis of pituitary adenomas from patients with CNC and acromegaly Patient
1 2 3 4 5 6 7 8
PRL (g/L)
Tumor size
Curea
NA
Macro
5.4 14.2 4.8 12.6
69.2 14.8 27 NA
211.5
41
38.6 54.4
Preoperative period
IGF-I* (nmol/L)
GH (g/L)
Presented with acromegaly 5 yr 1 yr 2 yr Presented with acromegaly Presented with acromegaly Presented with acromegly 1 yr
135.9
27
80.5 39.2 33 115.9
Adenoma staining
Hyperplasia
CGH changes
⫹
Yesb
Yes
⫹ NA ⫹ NA
⫺ NA ⫺ ⫹
NA NA Yesb Yesb
No No NA NA
NA
NA
NA
Yesb
NA
⫹
⫹
⫺
⫹
Yesb
NA
⫹
⫺
NA
NA
NA
No
GH
PRL
␣-sub
TSH
LH
No
⫹
⫹
NA
⫹
Micro Micro Micro Macro
Yes Yes Yes NA
⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹
⫹ NA ⫺ ⫹
126.1
Macro
No
⫹
⫹
8.3
12
Micro
Yes
⫹
4.7
13.3
Micro
Yes
⫹
Cure was defined as postoperative GH levels below 1 g/L, and normalization of the plasma IGF-I levels (IGF-I levels from all patients preoperatively were higher than the upper normal for the assay used and for the respective age group) and GH responses to oral glucose tolerance test (data not shown). Data are presented in Systeme International units (1 nmol/L ⫽ 7.649 ng/mL for IGF-I). b In these cases, more than one tumor was seen, in addition to the adenoma detected originally by magnetic resonance imaging. NA, Not available. CGH ⫽ comparative genomic hybridization; IGF-I ⫽ insulin-like growth factor type I, Macro, macroadenoma (greatest diameter over 1 cm); Micro, microadenoma (greatest diameter ⬍1 cm). a
also present in diffusely and rarely present cells of some adenomas and within foci of normal cells entrapped within the tumors (Table 2). ACTH and FSH staining, when obtained, was interpreted as derived from normal cells entrapped within the tumors or the hyperplasia (Fig. 1, 11 and 12). CGH analysis
CGH analysis of three tumors (cases 2, 3, and 8) showed no significant changes over normal DNA. In contrast, analysis of the most aggressive tumor, an invasive macroadenoma (case 1), showed multiple changes, including losses of chromosomal regions 6q, 7q, 11p, and 11q and gains of 1pter-p32, 2q35-qter, 9q33-qter, 12q24-qter, 16, 17, 19p, 20p, 20q, 22p, and 22q (Fig. 2). The greatest contiguous changes were losses of the long arm of chromosome 6 and the entire chromosome 11. Discussion
Acromegaly is usually characterized by a slow, progressive course. In CNC this course is even slower and acromegaly is often unmasked by adrenalectomy for primary pigmented nodular adrenocortical disease (28). In our prospective evaluation of patients with CNC, we have had the opportunity to observe the “development” of pituitary adenomas in four patients (cases 2, 3, 4, and 8). In these cases, biochemical abnormalities of GH and PRL secretion were present before radiological detection of a pituitary tumor (9, 10). The incidence of GH-producing pituitary tumors in CNC has been estimated at less than 10% (1, 6, 11), whereas GH “paradoxical” responses to various stimuli (such as to TRH) or IGF-I elevation, may be present in up to 80% of affected patients who have no detectable tumor (C.A. Stratakis, L.S. Kirschner, and J.A. Carney, unpublished data). The finding of probable hyperplasia in pituitary tissue from patients with CNC who underwent TSS for acromegaly is consistent with these clinical observations.
The GH-secreting adenoma in five of eight of these patients appeared to be surrounded by regions with expanded irregular reticulin structure, featuring GH, PRL, and occasionally ␣-subunit immunoreactive cells. These areas were shown to be identical by staining consecutive slides, because double immunostaining or electron microscopy were not available for analysis. It is noteworthy that in all patients multiple tumors were seen; the surface of the gland was covered with macroscopic tumors in at least four patients (patients 2, 4, 7, and 8; Table 2). In most of these cases, multiple tumors were identified microscopically, in addition to hyperplasia. PRL staining was not present in all the GHstained areas. This is consistent with finding PRL levels in the peripheral blood that were not markedly elevated in most patients with CNC (Table 2), in contrast to GH or IGF-I levels in the same patients. The genetic investigation complemented the above findings by suggesting that in its evolution, the largest and most aggressive tumor had accumulated a series of genetic changes; in contrast, the small adenomas had normal CGH results. These findings are in agreement with the hypothesis that pituitary tumors develop from clonal expansion of transformed somatic cells (29, 30). They are also consistent with observations in patients with MAS (14 –16) and some patients with MEN 1 (31). CNC and MAS are genetic conditions that share skin pigmentation abnormalities, adrenocortical hyperplasia, thyroid tumors, and even myxomas. However, in most tissues, the lesions are histologically and clinically different in the two conditions: skin lentigines and blue nevi vs. cafe´-au-lait spots, micronodular and pigmented dysplasia vs. adrenocortical macronodular hyperplasia, hormonally silent thyroid nodules or cancer vs. thyroid hormone hypersecretion, and skin vs. intramuscular myxomas (1, 6, 14, 32, 33). Mammosomatotroph hyperplasia may be the only lesion that is, in fact, common in CNC and MAS (34). Clinically, too, both share a “proacromegalic” state (35), which only rarely leads to the detection of an adenoma (10, 14 –16, 35). Similar long-standing somatotroph hyperplasia, which only occa-
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FIG. 1. Immunohistochemistry in pituitary tumors from patients with CNC and acromegaly. Images 1–3 are from case 1 (Table 2): 1, abnormally expanded and irregular reticulin pattern, consistent with adenohypopheseal cell hyperplasia (⫻100); 2, expanded reticulin pattern with beginning disruption (left and center) consistent with adenoma development (center) within hyperplasia (right) (⫻100), 3, disrupted reticulin pattern in the adenomatous tissue from the same patient (⫻40). Images 4 – 6 are from case 4 (Table 2): 4, hematoxylin and eosin staining of a zone of transition from tumorous (right) to normal pituitary tissue (left) (⫻40); 5, strong staining for GH in the pituitary adenoma of this patient (⫻40); 6, strong patchy staining for PRL in the same tumor (⫻40). Images 7–12 are from case 2 (Table 2). In this patient, areas of hyperplasia could not be seen in the small amount of excised tissue that was available for study. However, there were multiple tumors seen macroscopically; microscopically, there were different types of cells within the microadenoma: 7, junction of normal pituitary adenoma (center and left) and adenoma with larger cells (right) (⫻200); 8, uneven (strong and weak) staining for GH (⫻200); 9, nonhomogeneous (weak to strong) staining for PRL (⫻200); 10, brown cytoplasmic staining in many cells indicating positivity for ␣-subunit in the same tumor (⫻400); 11, immunostaining for ACTH showed no staining in the adenoma (left and center), whereas normal corticotrophs (right) stained appropriately (⫻200); 12, likewise, immunostaining for FSH was negative in adenomatous tissue (left and center) but positive in surrounding normal anterior pituitary tissue (right) (⫻200).
sionally leads to pituitary adenoma, has been seen in several other situations, albeit GHRH induced (36, 37). The genetic changes required for the formation of a pituitary adenoma in the background of benign hyperplasia are not known but seem to be multiple. As the present report has demonstrated, and other investigators have shown in tumors with Gs␣ mutations or allelic losses of the MEN 1 locus (38, 39), pituitary genetic changes tend to increase in number and significance in parallel with the clinical behavior
of the neoplasm (29, 30). Thus, pituitary tumorigenesis in CNC patients may follow the pattern of mutation accumulation that has been suggested for other neoplasms (40, 41). The extensive genetic instability of cells cultured from CNC tumors (17) suggests that secondary “hits” underlie tumor formation in CNC, the first “hit” being the germ line mutation. This corresponds to Knudson’s (42) hypothesis. The genes responsible for CNC seem to be involved in both loss and copy number gain or amplification (43). Thus, we spec-
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FIG. 2. A, Representative CGH image from the GH-producing macroadenoma shown in Fig. 1.1, 1.2, and 1.3. B, CGH profile averaged over at least eight metaphases per chromosome for the GHproducing tumor shown above (case 1, Tables 1 and 2). The arrows point to the main chromosomal defects, losses of 6q and whole chromosome 11, that were identified and are discussed in the text.
ulate that the germ line CNC mutation causes a predisposition toward other molecular events that are necessary for pituitary tumor formation in CNC patients. Acknowledgments We thank Drs. E. Oldfield (NINDS, NIH) and S. Marx (NIDDK, NIH) for excellent clinical services and useful discussions on pituitary tumors and for advice and guidance on multiple endocrine neoplasias, respectively. We owe immensely to Dr. David Katz, who as a staff pathologist at NCI, NIH, did some of the first readings of pituitary slides from patients with CNC and spent numerous hours with Dr. C. A. Stratakis over the microscope. We also thank the nursing and other support staff of NICHD, NIH, on the 8W and 9W wards for support of our research studies and help in the management of patients with CNC.
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