STUDIES ON ECTOPIC ACTH-PRODUCING TUMORS II. Clinical and Biochemical Features of 30 Cases
HIROO IMURA, * SHIGERU MATSUKURA,’ HIRONOSUKE YAMAMOTO,” YUKIO HIRATA, § YOSHIKATSU NAKAI,” JIRO ENDO, ** AKIRA TANAKA, ” AND MASUHISA NAKAMURA#
This report describes the clinical and biochemical features of 30 cases of ectopic ACTH-producing tumors diagnosed by the detection of ACTH in the tumor tissues. Several uncommon tumors, such as tumors of the esophagus, stomach, and larynx, were included in this series. None of the patients with bronchogenic carcinoma showed signs of classical Cushing’s syndrome, whereas 7 of the remaining 13 patients with other tumors were Cushingoid in appearance. Adrenocortical hyperfunction was present in 61% at the first ex- amination and developed during the course of the disease in 18% more. In the remaining patients (21%), adrenocortical function remained within normal limits. These results indicate that there exist ectopic ACTH-producing tumors without clinical and biochemical sequelae of excess hormone. In some of the tumor extracts studied, MSH and CRF-like activities and serotonin were detected. This suggests that multiple hormone production is not uncommon in ectopic ACTH-producing tumors.
Cancer 35:1430-1437, 1975.
T HE ASSOCIATION OF CUSHING’S SYNDROME with tumors of nonpituitary and non- adrenal origin has been occasionally re- ported since the original description of Brown.3 Meador et al.18 studied five patients with this syndrome and demonstrated the
From the Department of Medicine, Kobe University School of Medicine, Kobe, the Department of Medicine and Central Laboratory, Kyoto University School of Medicine, Kyoto, and Shionogi Research Laboratory, Osaka, Japan. Supported in part by research grants from the Ministry of Education, Japan.
* Professor of Medicine.
* Associate in Medicine.
* Assistant Professor of Medicine.
§ Fellow, Department of Medicine, Kobe University School of Medicine, Kobe.
Fellow, Department of Medicine, Kyoto University School of Medicine, Kyoto.
** Associate, Central Laboratory, Kyoto University School of Medicine.
** Chief, Endocrinolgy Section.
# Research Associate, Shionogi Research Laboratory, Osaka.
Address for reprints: Hiroo Imura, MD, Third Division, Dept. of Medicine, Kobe University School of Medicine, Kusunoki-cho, Ikuta-ku, Kobe, Japan.
The authors gratefully acknowledge Dr. Shichiro Ishikawa, National Cancer Center, Dr. Nobuaki Sasano, Tohoku University, and Dr. Osamu Midorikawa, Kyoto University, for valuable advice and help to this study. Thanks are also due to Drs. Shoji Hattori and Hiroshi Nishihara, Center for Adult Diseases, Osaka, for the supply of tumor tissues and measurement of serotonin.
Received for publication May 22, 1974.
presence of ACTH-like activity in tumor tissues. They postulated that these nonpituitary tumors elaborate ACTH-like materials, thus causing hyperadrenocorticism. Later, they referred to this syndrome as the ectopic ACTH-producing tumor. 12
With the progress of laboratory procedures in the past decade, an increasing number of patients with this syndrome has been reported, and many investigators have become aware of the presence of ectopic ACTH-producing tumors without clinical features of Cushing’s syndrome. However, the incidence and the pathogenesis of ectopic ACTH production are not clear at present.
In the past 8 years, we have detected ACTH activity in tumor tissues from 30 patients in our own and other institutions in Japan. The pre- sent communication summarizes clinical and laboratory data of these 30 patients, as well as the concentration of certain hormones in the tumor tissues.
METHODS
Clinical records and laboratory data such as blood sugar, serum electrolytes, urinary excre- tion of 17-hydroxycorticosteroids (17-OHCS) and 17-ketosteroids (17-KS), and the results of the dexamethasone suppression test, the ACTH
stimulation test, and the metyrapone test were sent by the physicians who cared for these patients. In 11 of 30 patients, plasma cortisol and urinary 17-OHCS and 17-KS were measured in our laboratory by competitive pro- tein binding assay,19 a modification of the method of Reddy et al.23 and the method of Drekter et al.,5 respectively.
Tumors obtained either at surgery or at autopsy within 12 hours of death were sent to our laboratory in a frozen state. A modification of the method of Payne et al.21 was used to ex- tract hormones from tumor tissue.8 This extract
(Extract I) was bioassayed either by the method of Lipscomb and Nelson14 or by the method of Vernikos-Danellis et al.27 Extract I from 7 of 30 tumors was tested for ACTH by radioim- munoassay according to the method of Imura et al.1º In the other 17 tumors, radioimmunoassay was performed by the method of Matsukura et al.17 after partial purification of Extract I.8
The melanocyte-stimulating hormone (MSH) activity of Extract I was determined in vivo in hypophysectomized African frogs, Xenopus laevis D.20 The in vitro pituitary incubation method was used to measure the activity of the
| Case | Sex | Age | Tumor | Cushing's syndrome | Chemical hyperadreno- corticism | Tumor ACTH bioassay mU/g |
|---|---|---|---|---|---|---|
| 1 | M | 62 | Oat-cell ca, bronchus | * + | 3.2 | |
| 2 | M | 53 | Oat-cell ca, bronchus | + | 0.02 | |
| 3 | M | 54 | Oat-cell ca, bronchus | + | 0.14 | |
| 4 | M | 59 | Oat-cell ca, bronchus | 1.6 | ||
| 5 | M | 45 | Oat-cell ca, bronchus | 1.7 | ||
| 6 | M | 58 | Oat-cell ca, bronchus | 0.12 | ||
| 7 | F | 68 | Oat-cell ca, bronchus | 0.06 | ||
| 8 | M | 66 | Oat-cell ca, bronchus | + | 0.18 | |
| 9 | M | 54 | Oat-cell ca, bronchus | + | 0.07 | |
| 10 | M | 34 | Oat-cell ca, bronchus | + | 2.2 | |
| 11 | M | 57 | Undifferentiated ca, | + | 0.17 | |
| bronchus | ||||||
| 12 | F | 63 | Undifferentiated ca, | + | 0.63 | |
| bronchus | ||||||
| 13 | M | 37 | Undifferentiated ca, | + | 0.43 | |
| bronchus | ||||||
| 14 | M | 63 | Adenocarcinoma, | + | 0.33 | |
| bronchus | ||||||
| 15 | M | 71 | Adenocarcinoma, | + | 18.4 | |
| bronchus | ||||||
| 16 | M | 57 | Adenocarcinoma, | 0.71 | ||
| bronchus | ||||||
| 17 | M | 68 | Squamous cell ca, bronchus | + | 0.06 | |
| 18 | M | 41 | Carcinoid, bronchus | + | + | 0.58 |
| 19 | F | 32 | Malignant epithelial | + | + | 0.45 |
| thymoma | ||||||
| 20 | F | 32 | Malignant epithelial | + | + | 0.33 |
| thymoma | ||||||
| 21 | M | 47 | Malignant epithelial thymoma | - | + | 0.02 |
| 22 | M | 34 | Malignant epithelial thymoma | + | + | 24.9 |
| 23 | F | 9 | Islet cell ca, pancreas | + | + | 0.10 |
| 24 | F | 33 | Islet cell ca, pancreas | + | + | 0.56 |
| 25 | M | 50 | Carcinoid, bronchus | + | 7.61 | |
| 26 | M | 62 | Oat-cell ca, esophagus | 3.2 | ||
| 27 | F | 57 | Oat-cell ca, esophagus | 0.08 | ||
| 28 | M | 60 | Carcinoid, esophagus | 0.73 | ||
| 29 | F | 51 | Carcinoid, stomach | + | + | <0.03+ |
| 30 | M | 66 | Squamous cell ca, larynx | + | 0.16 |
* - represents change of laboratory data.
Although bioassayable ACTH was not significant in this experiment, radioimmunoassayable ACTH was 115.9 ng/g tissue. This discrepancy was explained by the presence of biologically less active big ACTH, which was demon- strated by gel chromatography (unpublished observation).
corticotropin-releasing factor (CRF). Details of the method will be published elsewhere (in preparation). The tumor content of serotonin was determined by Dr. Nishihara of The Center for Adult Diseases, Osaka, by a modification of the method of Snyder et al. 25
Histologic examination was performed with routine hematoxylin and eosin stained sections in every case; in some cases special stains were used.
RESULTS
Histology of Tumors
The primary sites and histologic types of tumors are summarized in Table 1. Lung tumors predominated in the present series (19 of the 30 cases). Among these 19, there were 10 oat cell carcinomas, 3 undifferentiated carcinomas, 3 adenocarcinomas, 1 squamous cell carcinoma, and 2 bronchial carcinoids. Besides lung tumors, malignant epithelial thymoma, islet cell car- cinoma of the pancreas, and carcinoma of the es- ophagus were relatively common in the present series. A unique tumor in this series was squamous cell carcinoma of the larynx. 29 This patient, a 66-year-old man, underwent surgical excision of the larynx for squamous cell car- cinoma in 1971. One year later, a liver tumor was found in association with chemical hyperadrenocorticism. Postmortem examina- tion revealed metastasis of the laryngeal tumor to the liver and other organs. Bioassayable and immunoassayable ACTH as well as bio- assayable MSH were detected in the met- astatic tumor tissue.
Clinical Features
Classical features of Cushing’s syndrome, such as moon face, truncal obesity, hirsutism, acne, purple striae, and easy bruisability were noted in only 7 of the 30 patients, as shown in Table 1. None of the patients with lung cancer showed signs of Cushing’s syndrome, where- as 7 of the remaining 13 cases (54%) were Cushingoid in appearance. It is noteworthy, however, that truncal obesity, one of the most common symptoms in nonparaneoplastic Cushing’s syndrome, was absent in 2 of the 7 Cushingoid patients.
Skin pigmentation, edema, muscular weakness, and mental symptoms were docu- mented in 23%, 30%, 17%, and 30%, respectively. Hypertension, over 150 mm Hg systolic and 90 mm Hg diastolic, was noted in only 38% of the patients.
Routine Laboratory Examinations
Glycosuria was present in 32% of the cases, and fasting blood glucose levels over 120 mg/dl were documented in 38% of the patients tested (Fig. 1). A glucose tolerance test was performed in 14 patients and showed a diabetic pattern in 13 (93%). Hypokalemia, less than 3.5 meq/l, was recorded in at least one of repeated ex- aminations in 21 of 29 patients (Fig. 1).
Endocrinologic Examinations
Urinary excretion of 17-OHCS was deter- mined in 28 cases, and increased levels of more than 8 mg/day were recorded in 23. The
Fasting Blood Sugar
mg/dl
400-
300-
200
100
60
mEq/L
Serum K
5
4
3
2
1
Plasma Cortisol
Urinary 17-OHCS
Urinary 17-KS
Pl
4
mg/
4
M
F
day
50
100
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40
Î
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·1
1
remaining 5 patients showed urinary 17-OHCS levels less than 8 mg/day in one or repeated ex- aminations (Fig. 2). On the other hand, urinary 17-KS excretion was within normal limits or slightly subnormal in most patients, as shown in Fig. 2. Only 7 patients (2 with islet cell car- cinoma, 2 with malignant thymoma, 1 with bronchial carcinoid, 1 with lung cancer, and 1 with cancer of the larynx) showed increased levels. Five of these 7 patients had clinical features of Cushing’s syndrome.
Plasma cortisol levels were measured in 17 patients, either by fluorometry or by competitive protein binding assay. Increased morning levels were documented in 14 cases. Circadian rhythmicity of plasma cortisol was studied in 9 patients, by measuring morning (8 a.m.) and evening (6 p.m.) plasma cortisol levels. Only 1 patient, whose morning level was within normal limits, showed normal circadian rhythmicity. In the remaining 8 patients, whose morning cor- tisol levels were elevated, circadian rhythmicity of plasma cortisol was virtually absent.
Response of plasma cortisol or urinary 17- OHCS to ACTH was exaggerated in 3, almost normal in 3, and either limited or absent in the remaining 4 patients. The metyrapone test was performed in 7 patients. Three of them showed a normal response, and 4 did not respond. Oral administration of dexamethasone did not sup- press plasma cortisol or urinary 17-OHCS levels in 11 of 14 patients studied. Of the 3 suppressed patients, 2 showed normal adrenocortical func- tion at the time of examination. Another patient had elevated plasma cortisol and urinary 17- OHCS levels, which were suppressed by dex- amethasone, 10 mg daily.
In summary, adrenocortical function was studied before death in 28 of 30 patients, and
was within normal limits in 6 of them (Cases 4, 6, 16, 26, 27, and 28). Another 5 patients (Cases 1, 3, 10, 12, and 17) showed normal adrenocorti- cal function at the first examination, but hyperadrenocorticism developed during the course of the disease. Tables 2 and 3 show the results of endocrinologic studies in Cases 1 and 3, respectively. Case 1, a 62-year-old man, had normal plasma cortisol levels, which were sup- pressed by dexamethasone, and normal urinary 17-OHCS excretion in October, 1969. He was treated with cyclophosphamide with good results. In July, 1970, when the tumor relapsed, he had elevated plasma cortisol levels with lack of circadian rhythmicity and nonsuppressibility by dexamethasone, increased urinary excretion of free cortisol, and elevated plasma serotonin levels. Another example, Case 3, exhibited nor- mal adrenocortical function in May, 1970. He underwent pneumonectomy and subsequently received treatment with cyclophosphamide. In March, 1971, he showed profound hypokalemic alkalosis, with increased urinary excretion of 17- OHCS, and elevated plasma cortisol, ACTH,
| Oct. '69 | July '70 | |
|---|---|---|
| Plasma cortisol (µg/dl) | ||
| a.m. | 8.0 | 16.2 |
| p.m. | 6.0 | 14.8 |
| after dexamethasone | 1.5 | 14.0 |
| Urinary 17-OHCS (mg/day) | 7.8 | 7.5 |
| Urinary 17-KS (mg/day) | 2.2 | 3.7 |
| Urinary free cortisol (ug/day) | 156.2 | |
| Serum serotonin* (µg/1) | 268 |
* Serum serotonin was measured by Dr. H. Nishihara, Center for Adult Disease, Osaka.
| May '70 | Feb. '71 | March '71 | |
|---|---|---|---|
| Serum K (meq/1) | 4.3 | 4.1 | 2.3 |
| Urinary 17-OHCS (mg/day) | 5.5 | 25.5 | 57.0 |
| Urinary free cortisol (µg/day) | 23.4 | ||
| Plasma cortisol (ug/dl) | |||
| a.m. | 5.5 | 25.0 | |
| p.m. | 1.5 | ||
| after dexamethasone | 0.5 | ||
| Plasma ACTH (pg/ml) | 534 | ||
| Serum serotonin* (µg/1) | 102 | 79 | 509 |
* Serum serotonin was measured by Dr. H. Nishihara, Center for Adult Disease.
and serotonin levels (Table 4). In both patients, ACTH activity was detected in tumor tissues.
Hormones in Tumor Tissues
Bioassayable ACTH activity detected in tumor tissues is listed in Table 1. In all patients but one, tumor extracts showed significant adrenal steroidogenic activities. Figure 3 com- pares bioassayable and immunoassayable ACTH activities in tumor extracts in patients with hyperadrenocorticism to those in patients without chemical hyperadrenocorticism. Al- though patients lacking hyperadrenocorticism were few in number, there seems to be no signifi- cant difference in ACTH concentrations
between the two groups, and no difference was noted in the ACTH content of tumors of the lung and those of other organs (Fig. 3).
CRF and MSH activities and the serotonin content of tumor tissues were measured in cer- tain cases. As shown in Table 4, significant CRF activity was present in all cases studied. MSH activity not attributable to the intrinsic MSH activity of ACTH was detected in 5 of 8 tumors. The remaining 3 tumor extracts also showed some, but not significant, MSH activity. Serotonin was detected in significant amounts in the tissues of 4 of 6 patients. Thus, multiple ec- topic hormone production was observed in 9 of 11 tumor tissues in which more than two hor- mone assays were performed (Table 4).
DISCUSSION
The present studies support the previous observations2,9,13,14 that tumors of the lung, thymus, and pancreas are the main ectopic ACTH producers. However, the present series includes several uncommon tumors: three of the esophagus, a carcinoid tumor of the stomach, and a squamous cell carcinoma of the larynx. Lohrenz and Custer16 reported a case of squamous cell carcinoma of the esophagus with chemical hyperadrenocorticism, and demonstrated ACTH activity in liver me- tastases. Three cases of esophageal tumors in this series showed histologic findings diagnosed as either oat-cell carcinoma or carcinoid.
| Histology/Case | Tumor examined | MSH* | CRF+ | Serotonin ug/gr | |
|---|---|---|---|---|---|
| Oat cell ca | |||||
| 1 | Liver meta. | - | 0.26 | - | |
| 2 | Lung | 0.26ª | 7.4ª | +$ | |
| 3 | Lung | 50 | 0.13* | 8.8ª | + |
| 4 | Liver meta. | 1.33ª | + | ||
| 5 | Lung | 143 | >0.50* | 1.39* | + |
| 10 | Lung | 0.48* | + | ||
| Adenocarcinoma | |||||
| 16 | Lymph node meta. | - | - | ||
| Islet cell ca | |||||
| 24 | Pancreas | 421 | >0.70* | + | |
| Liver meta. | 507 | >0.23+ | + | ||
| Oat cell ca | |||||
| 26 | Liver meta. | - | 0.39* | + | |
| Squamous cell ca | |||||
| 30 | Liver meta. | 317 | + |
* MSH activity was expressed as ng human 8-MSH equivalent per g wet tissue.
CRF-like activity was estimated by adding tumor extract equivalent to 0.3 g wet tissue to the pituitary halves, and expressed as median eminence equivalent per flask.
* Significantly higher than in control tissues.
$ + represents multiple hormone production.
30
3000
20
2000
10
-1000
· Bioassayable ACTH (mU/gr.)
· Radioimmunoassayable ACTH (ng/gr. )
1
100
0.5
50
00
0.1
00
10
Lung Cancer Other Tumors
+
Hyperadrenocorticism
Adrenocortical function was within normal limits in these cases before death, although bioassayable and immunoassayable ACTH ac- tivities in the tumor tissues were positive at post- mortem examination. This suggests that ectopic ACTH production might be not uncommon in tumors of the esophagus, and would be detected if ACTH was measured more often.
Davis and Kennedy4 described a probable ACTH-producing carcinoid tumor of the stomach, although they did not measure tumor ACTH activity. Case 29 in this series showed classical features of Cushing’s syndrome, which were ameliorated by the removal of a sub- mucosal tumor of the stomach. The tumor showed the histologic features of carcinoid and contained ACTH, detected by radioimmuno- assay. This is perhaps the first definite case to be reported of carcinoid of the stomach with ectopic ACTH production.
One of the patients in this series had squamous cell carcinoma of the larynx and developed hyperadrenocorticism after the tumor had metastasized to the liver. Bioassayable and radioimmunoassayable ACTH as well as bioassayable MSH were detected in the metastatic tumor tissue. To our knowledge, there has been no report of squamous cell car- cinoma of the larynx with ectopic ACTH production.
After reviewing the pathology of reported cases, Azzopardi et al.1 postulated that the type of tumor had been erroneously diagnosed in some reports and that ectopic ACTH produc- tion occurs in only four types of tumors: oat-cell
carcinoma of the bronchus, endocrine tumors of foregut origin, pheochromocytoma and related tumors, and certain ovarian tumors. In the pres- ent series, there were five tumors which did not fit any of these categories; three adenocar- cinomas of the lung, and one squamous cell car- cinoma of the lung and one of the larynx. These tumors showed histologic features of ana- plastic carcinoma in some parts, but in other parts there were either typical acinar structures, different from the ribbon pattern or rosette formation of oat-cell carcinoma, or well- differentiated cornifying epithelial cells. Four of these five patients had chemical hyper- adrenocorticism. Sachs et al.24 reported a case of Cushing’s syndrome cured by the removal of a tumor of the lung. Histologic ex- amination revealed adenocarcinoma, consist- ing of tumor cells in acinar and trabecular ar- rangement. These observations suggest that squamous cell carcinoma or adenocarcinoma can elaborate ACTH, although less commonly than do oat-cell carcinomas or carcinoid tumors.
Ectopic production of ACTH by tumors does not always result in classical Cushing’s syn- drome. Only one of nine patients in a series of Friedman et al.6 and two of nine patients in a series of Liddle et al.11 were Cushingoid in appearance. In our observation, none of the patients with bronchogenic carcinoma showed classical Cushing’s syndrome, whereas it was present in about half the patients with other types of tumors. This suggests either that Cushing’s syndrome seldom develops in bronchogenic carcinoma, even in the presence of
remarkable hyperadrenocorticism, or that many cases lacking Cushing’s syndrome are overlooked in tumors other than bronchogenic carcinoma. It is possible that most patients with broncho- genic carcinoma do not develop Cushing’s syndrome, because of the brief duration of the disease or because of malnutrition. Further studies are required to solve this problem.
Since symptoms suggestive of ectopic ACTH production, such as skin pigmentation or profound muscular weakness, are present in less than 30% of the cases, laboratory examinations are required for the diagnosis of this syndrome. In contrast to the low incidence of hypokalemia in Cushing’s syndrome due to pituitary ACTH excess, more than 70% of the patients showed hypokalemia, less than 3.5 meq/l, in this series; values less than 3.0 meq/l were documented in 55%. Profound hypokalemia in patients with hyperadrenocorticism is suggestive of ectopic ACTH production.
Adrenocortical hyperfunction evaluated by urinary and plasma steroid levels was present in 61% of patients at the first examination. In 18%, hyperadrenocorticism developed during the course of the disease or at the time of relapse, although it was normal at the first examination. This suggests either that a specific cell line elaborating ACTH proliferated during the late stage of the disease, or that ACTH synthesized by tumor cells reached levels sufficient to cause adrenocortical stimulation as the tumor became enlarged. It is noteworthy that adrenocortical function remained within normal limits in about 21% of patients with ectopic ACTH-producing tumors. Ratcliffe et al.22 also reported that “con- trol tumors” contained radioimmunoassayable and bioassayable ACTH comparable to that in the tumors of patients with ectopic ACTH syn- drome. It is possible, therefore, that certain tumors synthesizing ACTH do not release it at a rate sufficient to cause clinical evidence of hyperadrenocorticism. An alternate explanation is that certain tumors elaborate immunological- ly active precursors of ACTH predominantly, which may be converted to the bioactive form during the process of extraction. Studies on the biosynthesis of ACTH by tumors may clarify this problem. In any case, these tumors may be designated as ectopic ACTH-producing tumors
but not as ectopic ACTH syndrome, since there is no associated hyperadrenocorticism. Ectopic hormone production not associated with clinical and biochemical manifestations seems to be more common than is generally recog- nized.
Weintraub and Rosen28 found human chorionic somatomammotropin in 11 out of 128 nontrophoblastic tumors. This is of particular significance since these placental hormones are not produced in nonpregnant women, and hence the possibility of adsorption of the hormone by tumor tissues can be excluded.
In the present experiments, we measured the MSH, CRF, and serotonin content of certain tumor extracts. Melanocyte-stimulating activity was demonstrated in all the tumors studied, and it was significantly higher than could be explain- ed by the intrinsic MSH activity of ACTH in five of the eight tumors examined. We also demonstrated, by gel chromatography, the presence of melanocyte-stimulating substances with molecular sizes corresponding to B-lipo- tropin, ß-MSH and a-MSH in some tumor tis- sues (unpublished observation). It seems pos- sible that MSH might be found more frequently if sensitive radioimmunoassay were used.
Significant CRF-like activity was found in all the tumors examined in this series. This tumor CRF-like activity cannot be accounted for by the intrinsic CRF activities of ACTH or any other known hormones (unpublished observation). Upton and Amatruda26 reported the presence of CRF-like activity in ectopic ACTH-producing tumors. It seems possible, therefore, that certain tumors elaborate CRF-like substance(s) different from any known substance, although its nature remains to be clarified.
It is well-known that ectopic ACTH- producing tumors occasionally elaborate hor- mones other than ACTH and MSH. In our observations, CRF-like substance and serotonin were produced in most of the tumors studied. In addition, antidiuretic hormone was produced in Case 12. It appears, therefore, that multiple hor- mone production is a common feature of the ec- topic ACTH-producing tumor, if asymptomatic hormone production is detected by the assay of tumor tissue.
ADDENDUM
Since this paper was submitted for publication, G. Gewirtz and R. S. Yalow (F. Clin. Invest. 53:1022-1033, 1974) have reported that 14 of 15 tissue extracts of lung carcinoma from patients without clinical manifestations of Cushing’s syndrome contained immunoreactive ACTH, and that the predominant form of tumor ACTH was biologically less active, big ACTH. These findings are in accordance with our observations.
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