A Patient with Preclinical Cushing’s Syndrome and Excessive DHEA-S Secretion Having Unilateral Adrenal Carcinoma and Contralateral Adenoma
SANAE MIDORIKAWA, SHIGEATSU HASHIMOTO, MINORU KURIKI, KEN KATOH,
TSUYOSHI WATANABE, HIRONOBU SASANO* AND TETSUO NISHIKAWA **
Department of Internal Medicine III, Fukushima Medical University, School of Medicine, Fukushima 960-1295,
*Department of Pathology, Tohoku University of School of Medicine, Sendai 980-8575, and
** Department of Medicine, Yokohama Rosai Hospital, Yokohama 222-0036, Japan
Abstract. We report a case of preclinical Cushing’s syndrome in a 54-year-old male associated with bilateral adrenocortical tumours. Physical findings and general laboratory data were unremarkable except for mild hypertension (158/90 mmHg) and impaired glucose tolerance. Endocrinological evaluation revealed the presence of autonomous cortisol secretion including unsuppressible serum cortisol by 8 mg dexamethasone test (11 ug/dl), high serum DHEA-S (3580 ng/ml, normal: 400-3500) and increased urinary 17-KS excretion (31.0-35.8 mg/day, normal: 5.8-21.3). CT scan demonstrated the presence of tumours in both adrenals and bilateral adrenalectomy was subsequently performed. Histological examination of the resected specimens revealed an adrenocortical carcinoma on the right side and an adenoma on the left side with noticeable cortical atrophy in non-neoplastic adrenals. Immunohistochemical study of steroidogenic enzymes demonstrated that all the steroidogenic enzymes involved in cortisol biosynthesis were expressed in both right and left adrenal tumours. Enzymatic activities of 21, 17a, 18, 118-hydroxylases were detected in both right and left adrenals except for the absence of 118-hydroxylase activity in the left adrenal adenoma. Results of in vitro tissue steroidogenesis examined in short-term tissue culture of the specimens revealed no significant differences between carcinoma and adenoma in cortisol production, but the production of adrenal androgens in carcinoma was significantly higher than that in adenoma, which may indicate the importance of evaluating adrenal androgen levels in patients with adrenocortical neoplasms.
Key words: Preclinical Cushing’s syndrome, Adrenal incidentaloma, Adrenocortical carcinoma, DHEA-S, Steroidogenesis
(Endocrine Journal 46: 59-66, 1999)
THE prevalence of computed tomography (CT), ultrasonography (US) and/or magnetic resonance imaging (MRI) in clinical practice nowadays has increased the incidence of detecting adrenal tumours in clinically asymptomatic patients.
Among these tumours, adrenal incidentalomas with autonomous cortisol secretion, but not associated with any specific signs of Cushing’s syndrome, have been proposed to be categorized as a specified clinical entity, i.e., preclinical or subclinical Cushing’s syndrome, by several groups of investigators [1-4]. But the natural course or clinical relevance of so-called “preclinical Cushing’s syndrome” has not been established [1, 5, 6], including whether or not preclinical Cushing’s syndrome is a precedent form of full blown Cushing’s syndrome, and whether or not
autonomous cortisol secretion exerts hazardous effects on the clinical course of these patients. Adrenocortical tumours associated with clinically apparent adrenal Cushing’s syndrome or preclinical Cushing’s syndrome usually occur as solitary neoplasm, whether adenoma or carcinoma. Bilateral adrenocortical tumours in these patients are extremely rare and no patient simultaneously discovered with adrenocortical carcinoma and contralateral adenoma have not been reported in the literature.
We report a case of preclinical Cushing’s syndrome associated with bilateral adrenocortical tumours, carcinoma and adenoma in the contralateral adrenal. We performed immuno- histochemical analysis of the steroidogenic enzymes, and in vitro studies of steroidogenic enzyme activities and of corticosteroid production of these adrenocortical lesions by means of short- term culture in order to study possible differences between the hormonal features of these bilateral adrenocortical tumours.
Materials and Methods
Patients
A 54-year-old Japanese man was referred to Fukushima Medical University, School of Medicine, Fukushima, Japan, for endocrinological evaluation of his bilateral adrenal tumours. Bilateral adrenal masses were recently detected by abdominal CT scan in the course of clinical evaluation of his gallstones. Physical examination revealed no evidence of central obesity, moon face, striae cutis, nor acne without mild hypertension (158/90 mmHg). Laboratory data demonstrated a white blood cell count of 5400 (eosinophil: 3%, neutrophil: 59%), serum potassium level of 3.7-4.3 mEq/l and fasting blood glucose level of 75 mg/dl. A 75 g oral glucose tolerance test demonstrated impaired glucose tolerance (blood glucose level 168 mg/dl, 120 min normal range: less than 110 mg/dl).
CT scan study revealed bilateral adrenal masses, 4.0 cm and 1.5 cm in greatest dimension in the right and left adrenals, respectively. Both tumours appeared well-encapsulated without irregular and heterogeneous surfaces (Fig. 1). MRI study also demonstrated bilateral adrenal masses. Adrenal
| I Urinary excretion | normal range | ||
|---|---|---|---|
| 17-KS | 31.0-35.8 | mg/day | (5.8-21.3) |
| 17-OHCS | 9.0-10.2 | mg/day | (2.7-9.8) |
| II Blood levels (0800 h) | |||
| ACTH | 3.1 | pg/ml | (6.1-55) |
| cortisol | 11 | µg/dl | (4.4-17.4) |
| 11-deoxycortisol | 1.04 | ng/ml | (0.04-1.16) |
| DHEA-S | 3580 | ng/ml | (400-3500) |
| DHEA | 1.9 | ng/ml | (1.2-7.5) |
| androstenedione | 3.2 | ng/ml | (0.5-2.4) |
| aldosterone | 89 | pg/ml | (25-160) |
| corticosterone | 2.43 | ng/ml | (0.38-8.42) |
| DOC | 0.07 | ng/ml | (0.08-0.28) |
| progesterone | 0.5 | ng/ml | (0-5.3) |
| 17-OH-progesterone | 0.8 | ng/ml | (0.05-3.7) |
| testosterone | 3.1 | ng/ml | (2.7-11) |
| PRA | 0.4 | ng/ml/h | (0.47-472) |
DHEA-S, dehydroepiandrosterone sulfate; DHEA, dehydroepiandrosterone; DOC, deoxycorticosterone; PRA, plasma renin activity.
scintillation scan with 131I-adosterol showed bilateral asymmetrical uptake of the tracer (right > left), both before and after 2 mg dexamethasone suppression for ten days.
Results of endocrinological examinations are summarized in Tables 1 and 2. They are summarized as follows; slight increase in urinary 17-OHCS excretion, great increase in urinary 17- KS excretion, a normal level of serum cortisol at
| dexamethasone (mg/day) | 0 | 2 | 8 |
|---|---|---|---|
| 17-KS (mg/day) | 33.4 -38.5 | 48.1-49.8 | 35.5-45.9 |
| 17-OHCS (mg/day) | 9.6-10.5 | 15.4-18.4 | 15.0-17.0 |
| ACTH (pg/ml) | 3.1 | 2.7 | 2.2 |
| cortisol (µg/dl) | 11 | 13 | 11 |
| 11-deoxycortisol (ng/ml) | 1.04 | 1.40 | 0.95 |
| DHEA-S (ng/ml) | 3580 | 4290 | 5030 |
| DHEA (ng/ml) | 1.9 | 2.0 | 2.9 |
| androstenedione (ng/ml) | 3.2 | 5.0 | 3.8 |
| corticosterone (ng/ml) | 2.43 | 3.54 | 2.27 |
| DOC (ng/ml) | 0.07 | 0.08 | 0.06 |
DHEA-S, dehydroepiandrosterone sulfate; DHEA, dehydroepiandrosterone; DOC, deoxycorticosterone. Oral administration of dexamethasone every 6 h, the blood samples were collected at 0800 h.
0800 h without a normal patterns of circadian rhythm, and a high serum level of DHEA-S. Dexamethasone suppression test was performed by oral administration of 0.5 mg dexamethasone every 6 h for 2 days and 2 mg every 6 h for 2 days. Plasma cortisol, serum DHEA-S and other steroid hormone levels were measured at 0800 h on the second day of the administration and urinary excretion of 17-OHCS and 17-KS was measured every day. Insufficient suppression of these steroids was observed (Table 3). The release of plasma cortisol to ACTH was within normal limits. Selective venous sampling in the right and left adrenal veins was attempted but satisfactory results could not be obtained. Surgical resection of the bilateral adrenal glands was performed. The right and left adrenals weighted 45 g and 13 g, and appeared brown to yellow and yellow on their cut surface, respectively. Both tumours were well- encapsulated and well circumscribed. Marked cortical atrophy was detected in both non-neoplastic adrenals. Replacement therapy was done first by
intravenous administration of 50-100 mg of water soluble hydrocortisone during and after the operation, followed by continuous oral administration of 20-40 mg hydrocortisone. General scintillation scan with 131I-adosterol performed on the 30th day after the operation demonstrated no abnormal accumulation of the tracer in the liver, lungs or other organs. Op’DDD (mitotane) of 3.0 g/day was administered for 18 months after the operation as an adjuvant chemotherapy for his adrenocortical carcinoma. He is well without recurrence or metastasis in the 18 months since the operation.
In vitro steroidogenesis
Details of the methods have been previously described [7, 8]. The adrenal tumour specimens were divided to tumourous and non-tumourous regions, each cut into small pieces with scissors. These tissues were preincubated for 1 h in Krebs- Ringer bicarbonate medium containing 0.2% glucose under a 95% O2-5% CO2 atmosphere at 37 ℃ with continuous shaking. The media were subsequently replaced with 5 ml of the same solution, containing test agents such as 10-8 M of ACTH, and then further incubated for 2 h. After incubation, levels of various steroids in the medium were determined with a specific radioimmunoassay (RIA) kit; cortisol, dehydroepiandrosterone sulfate DHEA-S), dehydroepiandrosterone(DHEA), androstenedione and aldosterone concentrations were determined with a SPAC-S Cortisol kit (Daiichi Isotope Co. Ltd., Tokyo, Japan), DHEA-S, DHEA and androstenedione obtained from RIA kits of Diagnostic System Laboratories, USA, and an aldosterone RIA kit II (Dainabot Co. Ltd., Tokyo, Japan). 17a-Hydroxyprogesterone and proges- terone concentrations were measured with a RIA kit (Japan DPC Co. Ltd., Tokyo, Japan). The amount
| Daily profile of ACTH and cortisol | ||||||
|---|---|---|---|---|---|---|
| clock | 0800 h | 1200 h | 1600 h | 2000 h | 2400 h | 0400 h |
| ACTH (pg/ml) | 4.7 | 4.0 | 4.3 | 12 | 4.1 | 4.7 |
| cortisol (µg/dl) | 9.8 | 6.9 | 12.0 | 10 | 6.0 | 10 |
| ACTH infusion test | ||||||
| time (min) | 0 | 15 | 30 | 60 | 90 | 120 |
| cortisol (µg/dl) | 9.0 | 26.0 | 29.0 | 27.0 | 27.0 | 25.0 |
of each steroid hormone produced was calculated per unit of tissue weight (ng or ug).
Statistical analysis was performed by paired or unpaired Student’s t test and a P value less than 0.05 was considered significant.
Immunohistochemical study
Immunohistochemical studies of steroidogenic enzymes, including cholesterol side chain cleavage (P450scc), 3-hydroxysteroid dehydrogenase (3B- HSD), 21-hydroxylase (P450c21), 17a-hydroxylase (P45017a), 11ß-hydroxylase (P45011B), and DHEA- ST were performed with formalin-fixed, paraffin-embedded serial sections of the tumours by the biotin-strept-avidin amplified method and Histofine immunostaining system (Nichirei Co. Ltd., Tokyo, Japan). Immunostaining procedures and the characteristics of primary antibodies used in this study have been described previously by the authors [9, 10]. Phosphate buffered saline (0.01 mol/l) and normal rabbit IgG were used instead of primary antibodies as negative controls.
Biochemical activities of steroidogenic enzymes
The activities of 21-hydroxylase (P450c21), 17- hydroxylase (P450 17a), 11-hydroxylase (P45011฿) and 18-hydroxylase were measured as described previously by the authors [9-11] in microsomal or mitochondrial fraction obtained from both tumours.
Results
Histopathology
The right tumour was predominantly composed of compact cells with abundant eosinophilic cytoplasm (Fig. 2A). Among nine histopathological criteria of adrenocortical malignancy proposed by Weiss [12], four criteria including compact cytoplasm, diffuse arrangement, atypical nuclei and necrosis were positive. This tumour was therefore histopathologically diagnosed as adrenocortical carcinoma. The left tumour was predominantly
A
B
A
C
P<0.05
p<0.05
15.0-
NS
30
P<0.05
cortisol (¿4g/g tissue/hr)
DHEA-S (ng/g tissue/hr)
10.0
NS
20
p<0.05
5.0
10
not detectable
not detectable
rt. ad. carc.
It. ad. aden.
rt. ad. carc.
It. ad. aden.
rt. ad. carc.
It. ad. aden.
It. ad. aden.
rt. ad. carc. ACTH administration
control
ACTH administration
control
p<0.01
B
D
P<0.01
androstendione (ng/g tissue/hr)
1500
p<0.05
DHEA (ng/g tissue/hr)
1500
1000
1000-
P<0.01
P<0.01
500
500
rt. ad. carc.
It. ad. aden.
rt. ad. carc.
It. ad. aden.
rt. ad. carc.
It. ad. aden.
rt. ad. carc. ACTH administration
It. ad. aden.
control
ACTH administration
control
composed of clear cells (Fig. 2B). Because none of the above mentioned criteria of Weiss was negative in the left tumour, it was histopathologically diagnosed as adrenocortical adenoma. Marked cortical atrophy was observed in the adjacent non- neoplastic adrenal on both right and left sides.
Short-term culture of tissue slices
Results of in vitro steroidogenesis in the adrenal tumours performed in short-term culture of tissue slices are summarized in Fig. 3. There was much more production of adrenal androgens, such as DHEA-S, DHEA, and androstenedione in the adrenal carcinoma than in the adrenal adenoma under both basal and ACTH-stimulated conditions
(Fig. 3). In contrast, there were no differences between carcinoma and adenoma specimens in the synthesis of cortisol (Fig. 3A), 11-deoxycortisol, 17- OH-progesterone and progesterone (data not shown).
Immunohistochemistry
Results of immunohistochemistry of steroidogenic enzymes are summarized in Table 4. In adenoma, immunoreactivity of 3B-HSD, P450scc, P450c21, P450c11 and P450c17 were detected in tumour cells. In carcinoma, P450c21 and P450scc were relatively diffusely expressed but immuno- reactivity of 3B-HSD, P450c11 and P450c17 were only focally observed in carcinoma cells. DHEA-
| rt. | adrenal carcinoma | lt. adrenal adenoma |
|---|---|---|
| P450scc | + | + |
| 3B-HSD | + | + |
| P450c21 | f+ | + |
| P45017a | f+ | + |
| P45011B | f+ | + |
| DHEA-ST | f+ | - |
-, negative; +, positive; f+, focally positive.
ST was focally positive in the carcinoma (Fig. 4A), but negative in the adenoma (Fig. 4B). None of the steroidogenic enzymes including DHEA-ST was expressed in the atrophic cortex in either the right or the left adrenal.
Biochemical activities of steroidogenic enzymes
Results were summarized in Table 5. 21- Hydroxylase (P450c21), 17a-hydroxylase (P45017a), and 18-hydroxylase activities were detected in both the right and left adrenal tumours, while 11ß- hydroxylase (P45011B) was detected only in the
carcinoma. 21-Hydroxylase activity was stronger in adenoma than in carcinoma but 17a-hydroxylase activity was stronger in carcinoma than in adenoma.
Discussion
Cases of full blown Cushing’s syndrome due to bilateral adrenal tumours have been reported [13- 19], but not in those with preclinical Cushing’s syndrome as in this case. In addition, the case reported here is also the first reported case of unilateral adrenocortical carcinoma associated with contralateral adenoma. In the case of bilateral adrenocortical lesions, it is important to determine which adrenals may contribute to hormonal abnormalities. The results of adrenal scintillation scan with 131I-adosterol showed the production of steroid hormone on both sides (right > left). Selective venous sampling generally provides important information on the laterality of steroid secretion, but we could not obtain satisfactory results despite several attempts. We therefore performed immunohistochemical analysis of the steroidogenic enzymes, in vitro studies of
A
B
| enzymes | normal range | rt. carcinoma | lt. adenoma |
|---|---|---|---|
| 21-hydroxylase (n moles/mg protein/2 min) | 7.5 ± 2.5 | 1.6 | 5.4 |
| 17-hydroxylase | 1.4 | 3.99 | 1.92 |
| (n moles/mg protein/2 min) | |||
| 18-hydroxylase | 1.4 | 3.6 | 3.7 |
| (aldosterone formed ng/mg protein/2 min) | |||
| 11ß-hydroxylase (11-OHCS formed µg/mg protein/7.5 min) | 5.7 ± 1.9 | 3.5 | not detectable |
steroidogenic enzymes activities and analysis of in vitro corticosteroid production by short-term tissue culture in order to examine the possible differences between these bilateral adrenocortical tumours in endocrinological features.
Results of studies of in vitro steroidogenesis in these two tumours demonstrated that cortisol production per gram wet tissue was similar in the two. It is therefore suggested that relatively autonomous production of cortisol inducing preclinical Cushing’s syndrome seems to originate in the tumours on both sides, although there was difference between the degree of secretion of cortisol in the right and left adrenal tumours in vivo because of the difference in the size. Results of immunohistochemistry of steroidogenic enzymes are also consistent with those of in vitro tissue culture studies. The absence of 11ß-hydroxylase activity in the adenoma may be due to heterogeneity of the specimens or others, but it awaits further investigations for classification. The in vitro studies of steroidogenic enzymes demonstrated that 21-hydroxylase activity was lower in the carcinoma tissue than in the adenoma tissue. The experiments on organ cultures demonstrated that adrenal androgens including androstenedione, DHEA, and DHEA-S, were produced in greater quantity in the carcinoma tissue than in the adenoma tissue. These data suggest that the carcinoma tissue rather than the adenoma tissue may predominantly synthesize adrenal androgens because of the relative deficiency of 21- hydroxylase and well-maintained activity of 17-hydroxylase in the carcinoma tissue. Moreover, the carcinoma secreted significantly larger amounts of adrenal androgens in vitro than the adenoma and expressed DHEA-ST, which is consistent with previously reported endocrinological features of
adrenocortical carcinomas [9, 20]. The present experiments demonstrated that ACTH increased cortisol production by the carcinoma tissue, although there were several reports describing unresponsiveness of cortisol production to ACTH stimulation in human adrenocortical cancers [21]. Benign tumours are believed to be responsive to ACTH. It is therefore speculated that the present case of carcinoma is of low grade malignancy, and the patient is still alive without any metastasis so far. Discrepancy observed among steroidogenic enzyme activities, immunoreactivity and in vitro steroid production in short-term culture, especially in adrenocortical carcinoma, is considered to reflect disorganized steroidogenesis associated with the kind of adrenocortical malignancy reported by Sasano et al. [9], but further experiments are needed.
High serum adrenal androgens, such as DHEA- S and increased excretion of urinary 17-KS have been proposed by several groups to indicate the presence of adrenocortical malignancy [21, 22]. In this case, adrenocortical carcinoma secreted significantly larger amounts of adrenal androgens than adenoma in short-term culture of tissue slices. These adrenal androgens, especially DHEA-S, are considered to result in a high serum androgen level. Adrenalectomy is therefore clinically recommended in these cases of adrenocortical tumours with high adrenal androgen concentration, even in bilateral neoplasms as detected in this case.
Acknowledgement
The author thanks Ms. Atsuko Hashimoto for technical assistant.
References
1. Giangiacomo O, Massimo T, Giorgio B, Gianpaolo M, Anna AL, Alessandro P, Piero P, Alberto A (1994) Endocrine evaluation of incidentally discovered adrenal masses (Incidentalomas). J Clin Endocrinol Metab 79: 1532-1539.
2. Robert HC, Pamela JS, Gary GW (1994) Subclinical hormone secretion by incidentally discovered adrenal masses. Arch Surgery 129: 291-296.
3. Harold NR, Stephen LS (1992) Subtle glucocorticoid excesses in patients with adrenal incidentaloma. Am J Med 92: 213-216.
4. Martin R, Joachim N, Gabriel PK, Wolfgang S, Bruno A, Werner W (1992) Preclinical Cushing’s syndrome in adrenal “Incidentaloma”: Comparison with adrenal Cushing’s syndrome. J Clin Endocrinol Metab 75: 826-832.
5. Miyamori I, Iki K, Takeda R (1994) Preclinical Cushing’s syndrome: Report of a case and the review of the literature. Folia Endocrinol Japon 70: 25-30 (In Japanese).
6. Reincke M, Nieke J, Krestin GP, Saeger W, Allolio B, Winkelmann W (1992) Preclinical Cushing’s syndrome in adrenal “incidentalomas”: Comparison with adrenal Cushing’s syndrome. J Clin Endocrinol Metab 75: 826-832.
7. Ojima M (1978) In vivo and in vitro studies on steroid production Aldosteronoma. Folia Endocrinol Japon 54: 246-254 (In Japanese).
8. Saruta T, Robert C, Norman MK (1972) Adrenocortical steroidogenesis: Studies on the mechanism of action of angiotensin and electrolytes. J Clin Invest 51: 2239-2245.
9. Sasano H, Suzuki T, Nagura H, Nishikawa T (1993) Steroidogenesis in human adrenocortical carcinoma. Human Pathology 24: 397-404.
10. Sasano H (1994) Localization of steroidogenic enzymes in adrenal cortex and its disorder. Endocr J 41: 471-482.
11. Nagasaka S, Kubota K, Motegi T, Hayashi E, Ohta M, Takahashi K, Takahashi T, Iwasaki Y, Kolke M, Nishikawa T, Sasano H, Murakami T (1996) A case of silent 21-hydroxylase deficiency with persistent adrenal insufficiency after removal of an adrenal incidentaloma. Clin Endocrinol 44: 111-116.
12. Weiss LM (1984) Comparative histologic study of 43 metastasizing and non metastasizing adrenocortical tumors. Am J Surg Path 8: 163-169.
13. Iwase K, Nagasaka A, Tsujimura T, Inagaki A, Nakai A, Masunaga R, Kato S, Miura K (1994) Cushing’s syndrome with cortisol hypersecretion from one of bilateral adrenocortical adenoma: Report of a case. Jpn J Surg 24: 538-543.
14. Aiba M, Kawakami M, Ito Y, Fujimoto Y, Suda T, Demura H (1992) Bilateral adrenocortical adenomas causing Cushing’s syndrome. Arch Pathol Lab Med 116: 146-150.
15. Zeuger MA, Nieman LK, Culter GB, Chrousos GP, Doppman JL, Travis WD, Norton JA (1991) Primary bilateral adrenocortical causes of Cushing’s syndrome. Surgery 110: 1106-1115.
16. Mimou N, Sakato S, Nakabayashi H, Saito Z, Takeda R, Matsubara F (1985) Cushing’s syndrome associated with bilateral adrenal adenomas. Acta Endocrinol 108: 245-254.
17. Kato S, Masunaga R, Kawabe T, Nagasaka A, Miyamoto T, Itoh M, Nakai A, Iwase K, Tsujimura T, Ohtani S, Inagaki A, Miura K, Chikamatsu H, Hishide H, Mizuno Y (1992) Cushing’s syndrome induced by hypersecretion of cortisol from only one of bilateral adrenocortical tumors. Metabolism 41: 260-263.
18. Satoh K, Miyagata S, Harata T, Nishizawa O, Tsuchida S (1988) A case report of Cushing’s syndrome due to bilateral adrenal adenomas. Folia Urologica Japonica 79: 1463-1468 (In Japanese).
9. Milton DG, Brahm S, Isaac RF, Robert LB, Melvyn K, Michael KM, Norman WT, Jeffery AS (1995) Scintigraphy of discovered bilateral adrenal masses. Eur J Nuclear Medicine 22: 315-321.
20. Sasano H, Sato F, Shizawa S, Nagura H, Michael W, Coughtrie H (1995) Immunolocalization of dehydroepiandrosterone sulfotransferase in normal and pathologic human adrenal gland. Mordern Pathology 8: 891-896.
21. Yoshida A, Nishikawa T, Tamura Y, Yoshida S (1986) Evidence for decreased activity of guanine nucleotide binding protein in adenylate cyclase of cell membranes in human ACTH-unresponsive adrenocortical carcinoma. Endocrinol Japon 33: 891- 899.
22. 2. Margioris AN, Chrousos GP (1990) Cushing’s syndrome: Diagnostic evaluation. In: Biglieri EG, Merby JC (eds) Endocrine Hypertension. Raven Press, New York, 99.