American Journal of Pathology, Vol. 138, No. 1, January 1991 Copyright ( American Association of Pathologists

Immunoreactivity and Receptor Expression of Insulinlike Growth Factor I and Insulin in Human Adrenal Tumors An Immunohistochemical Study of 94 Cases

Takihiro Kamio, Kazuto Shigematsu, Kioko Kawai, and Hideo Tsuchiyama From the Second Department of Pathology, Nagasaki University School of Medicine, Nagasaki, Japan

Using immunoperoxidase methods, 94 human adrenal tumors were examined for evidence of immunoreactivity and receptor expression of insulinlike growth factor I (IGF-I) and insulin. The frequency of IGF-I in adrenocortical carcinomas was significantly higher than that in adenomas of the adrenal glands. The adrenocortical carcinomas showed strong intensity of staining for IGF-I, IGFI receptors, and insulin receptors. A significant correlation between immunoreactivity and receptor expression ofboth IGF-I and insulin was found only in the adrenocortical carcinomas. The adrenocortical adenomas with Cushing's syndrome and pheochromocytomas, more than adrenocortical adenomas with Conn's syndrome, also stained strongly for insulin receptors. Thus the IGF-I and insulin probably play a role in the growth of adrenocortical carcinoma tissues, possibly through autocrine mechanisms. The expression of insulin receptors in adrenocortical adenomas in the presence of Cushing's syndrome and pheochromocytomas may be associated withfunctions. (Am JPathol 1991, 138:83-91)

Growth factors seem to be involved in the transformation or proliferation of neoplastic cells.'-6 Insulinlike growth factors (IGFs) are homologous to the amino acid sequences of proinsulin.78 Receptors of insulinlike growth factor (IGF-1), one form of IGFs, are structually similar to insulin receptors.9'10 In addition, each peptide can bind to its own receptor and, to a lesser extent, to the other receptor.9'10 The production of IGF-I-like peptides and the enhancement of IGF-I receptors were noted in human neoplastic cells.2'5'51 -13

Many factors have been proposed to stimulate adrenocortical cell proliferation."4 Insulinlike growth factor I and insulin also regulate growth of the adrenal gland as well as maintain specific adrenal cell functions.15-17 Insulinlike growth factor immunoreactivity is detectable in the adrenal gland.1618 In addition, the existence of distinct receptors for both peptides has been reported.16'7 '920 Little is known of the activity of IGF-I and insulin in adrenocortical carcinomas and adenomas. In rat and human pheochromocytoma cells, insulin induces a differentiation or increase in the secretion of catecholamines.2123 Most recently, we obtained evidence that epidermal growth factor receptors (EGFr) with the intracellular tyrosin kinase domain24 are expressed in adrenocortical carcinomas, at a high frequency.25 Like EGFr, GE-I and insulin receptors also display a tyrosin kinase activity,'10'2627 followed by RNA synthesis and gene expression.28 Furthermore EGF may synergize with IGF-I to promote the growth of BALB/ c-3T3 cells, a cell line of mouse fibroblast.29 Hence the presence of IGF-I and insulin and their receptors must be investigated to elucidate mechanisms of cell growth and steroidogenesis in adrenal tumors. In the present study, we used an immunohistochemical method to determine whether or not there is receptor expression as well as immunoreactivity for IGF-I and insulin in adrenal tumors, including adrenocortical carcinomas obtained at autopsy, and adrenocortical adenomas and pheochromocytomas obtained during surgery.

Materials and Methods Tissue Samples Ninety-eight cases of adrenocortical carcinomas listed in the annual report of Pathological Autopsy Cases in Japan Supported in part by a grant from the Graduates' Association of Nagasaki University School of Medicine (to T. Kamio, 1989) and a Grant-in-Aid for Scientific Research from the Ministry of Japan (to K. Shigematsu, 1988 and 1989).

Accepted for publication August 28, 1990. Address reprnt requests to Kazuto Shigematsu, MD, PhD, the Second Department of Pathology, Nagasaki University School of Medicine, 12-4 Sakamoto-machi, Nagasaki 852, Japan.

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between 1965 and 1982 were collected. To avoid overt autolysis, great deterioration of immunoreactivity or receptor protein after death, paraffin blocks of 64 tissues obtained at autopsy within 3 hours after death'13,2030 were used for the immunohistochemical study. Twenty-three cases of adrenocortical adenomas (nine of Cushing's syndrome, 11 of Conn's syndrome, and three nonfunctioning) and seven pheochromocytomas were selected from 89 adrenal tumors filed in the Second Department of Pathology, Nagasaki University School of Medicine. The adrenal tumors were diagnosed according to the rules of the World Health Organization31 and the Armed Forces Institute of Pathology.32 Furthermore, the adrenocortical carcinomas examined were histologically graded on the basis of how closely the cells resembled normal cortical cells; Gl, well-differentiated type; G2, moderately differentiated type; and G3, poorly differentiated type.25'3

Immunohistochemistry Serial 5-,u-thick sections taken from all the paraffin blocks were stained for immunoreactivity and receptors of IGF-I

and insulin. Sections adjacent to the related tissue sections used for the immunostaining were stained with hematoxylin and eosin (H&E). After treatment with normal goat or horse serum for 60 minutes, sections were incubated with the primary antibody at the indicated dilutions: rabbit anti-human polyclonal IGF-I serum 1/100 (amino acids 1 to 70, IGF-I; KabiGen AB, Stockholm, Sweden), rabbit anti-human IGF-I receptor serum 1/50 (Upstate Biotechnology Inc, Lake Placid, NY), mouse anti-porcine monoclonal insulin serum 1/200 (Sekisui Biochemical Co., Osaka, Japan), and mouse anti-human monoclonal insulin receptor serum 1/50 (Cosmo Bio Co., Tokyo, Japan). The sections were incubated for 48 hours at 4°C for IGF-I receptors, for 60 minutes at room temperature for IGF-I and insulin receptors, and overnight at 4°C for insulin. After incubation, the tissue sections were washed three times (30 minutes each) with phosphate-buffered saline (PBS), pH 7.4. Localization of immunostaining was demonstrated by the avidin-biotin peroxidase complex method, using Vectastain ABC kits (Vector Laboratories, Burlingame, CA). Paraffin blocks of human squamous cell carcinoma of lung, placental, and pancreatic tissues obtained at surgery were used as positive controls.'1334 35 For the negative controls, tissues were preincubated with the IGF-I antibody with an

4.~~~~~~~~~~~~~~~~~~~~g

Figure 1. Immunostaining in positive control sections. Immunostaining of human squamous cell carcinoma of the lung with antiIGF-I serum (a), human placental villi with anti-IGF-I receptor (b), and anti-insulin receptor (c) sera and islet cells of human pancreas with anti-insulin serum (d) (magnification X350).

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excess of unlabeled IGF-I (Peninsula Laboratories), and by applying an irrelevant rabbit or mouse immunoglobulin serum instead of the IGF-I receptor, insulin, and insulin receptor antibodies. Several cases of adrenocortical adenomas were examined for a comparison of immunocytochemical staining in paraffin and fresh frozen sections.

Data Analysis The data obtained were expressed as scores ranging from 0 to 1 (1% to 10% positive cells), to 2 (10% to 50% positive cells), to 3 (50% to 100% positive cells), according to the ratio of positive cells in the tumors.253 Differences were analyzed by Chi-square test and Kendall's rank correlation test (Kendall's tau).

Results Human squamous cell carcinoma tissues were used for positive controls of IGF-1, whereas human placental tissues for receptors of IGF-I and insulin and human pancreatic tissues for insulin were examined. Insulinlike growth factor I immunostaining was observed in the carcinoma cells (Figure 1 a), columnar epithelium, cartilage, and pulmonary vessels. Immunostaining for receptors of IGF-I and insulin was evident in the placental chorionic villi (Figure 1 b, c). Islet cells of the pancreas contained insulin immunoreactivity (Figure 1 d). With regard to assessments of tissue preparations, a comparison was made of immunocytochemical staining in paraffin and fresh frozen sections. Although the immunohistochemical procedure used for the paraffin sections provided less intense peroxidase reactions than seen in the fresh frozen sections, there was no significant difference in the ratio of positive cells between both these preparations, when the tissue sections were incubated under the conditions described in the Materials and Methods. There was no evidence of positive immunostaining in the negative control sections. According to the differentiation, the adrenocortical carcinomas were classified into the Gl group (16 cases), G2 group (33 cases), and G3 group (15 cases) (Figure 2). As shown in Table 1, 16 of the 64 patients had functioning adrenocortical carcinomas: 13 with Cushing's syndrome (seven cases; plus virilization, six cases; unknown symptom of virilism), one with Conn's syndrome, and two with adrenogenital syndrome. Twenty-nine were clinically nonfunctioning and in the remaining 19, the function or lack of it in adrenocortical carcinomas was unknown. The adrenocortical adenomas examined never contained necrosis, mitosis, or vascular or capsular invasion.

Figure 2. Histologic grading ofadrenocortical carcinomas; Gl (a), G2 (b), and G3 (c) (magnification X 150).

In the non-neoplastic adrenal gland, strong immunostaining for IGF-I was observed in the zona reticularis, stained cells were scattered among adrenal medulla tissues, and the immunostaining in zona glomerulosa and zona fasciculata cells was minimal. The IGF-I receptors were prominantly localized in the zona reticularis and zona glomerulosa; the medulla demonstrated minimal staining. Conversely the binding of insulin receptor antiserum showed a prominent labeling of the inner zona fasiculata to the zona reticularis cells; the zona glomerulosa, outer zona fasciculata, and medulla showed slightly less immunoreactivity. The distribution of insulin was almost same

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Table 1. Immunoreactivities and Receptor Expressions for IGF-I and Insulin in Adrenal Tumors Positivity (%) Histologic types

Adrenocortical Carcinoma Cushing's syndrome Conn's syndrome Adrenogenital syndrome Nonfunctioning Unknown Adrenocortical Adenoma Cushing's syndrome Conn's syndrome Nonfunctioning Pheochromocytoma

No. of cases 64 13 1 2 29 19 23

9 11 3 7

IGF-I 58 (90.6) 11 1 2 27 17 11 (47.8)* 5 5 1 4 (57.1)t

IGF-I receptor

Insulin

Insulin receptor

60 (93.8) 12 1 2 26 19 22 (95.7) 9 10 3 5 (71.4)

55 (85.9) 8 1 2 26 18 16 (69.6) 7 7 2 5 (71.4)

58 (90.6) 13 1 2 25 17 23 (100) 9 11 3 7 (100)

t X2 = 11.898 (carcinomas vs. adenomas); P < 0.01.

t x2 = 16.369 (carcinomas vs. pheochromocytomas); P < 0.01.

as that of insulin receptors, but the zona glomerulosa also contained a moderate immunoreactivity. The immunoreactivity of IGE-I and insulin took the form of a fine granular pattern in the cytoplasm of the tumor cells (Figures 3a, 4a). The immunoreaction for receptors of IGF-I and insulin was limited to the cytoplasm (Figures 3b, 4b) and cell membrane (Figures 3c, 4c).

The results of staining for IGF-1, IGF-I receptors, insulin, and insulin receptors in 94 adrenal tumors are summarized in Table 1. Of the 64 cases of adrenocortical carcinomas, 58 (90.6%) showed IGF-I immunoreactivity. The frequency of IGF-I immunostaining in the adrenocortical carcinomas was significantly higher than in the adrenocortical adenomas (11 of 23 cases, 47.8%) and pheochromocytomas

Figure 3. IGF-I (a) and IGF-I receptor(b andc) immunohistochemical staining withbematoxylin counterstaining in adrenocortical carcinoma. d shows an alternate section of(a), which illustrates prevention of stainingfollowingpreincubation ofIGF-I antiserum with an excess of immunogen peptide. Arrows show immunoreactive cells (magnification X350).

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Figure 4. Immunohistochemical stainingfor insulin (a) and insulin receptors (b and c) with hematoxylin counterstaining in adrenocortical carcinoma. Immunoreactivity of insulin receptors concentrates in the cytoplasm (b) and cell membrane (c). d indicates a negative control. Arrows show immunoreactive cells (magnification X350).

(four of seven cases, 57.1%) (P < 0.01). There was no difference in the frequency of staining for IGF-I receptors, as expressed in 93.8% of adrenocortical carcinomas, 95.7% of adrenocortical adenomas, and 71.4% of pheochromocytomas. The incidences of insulin in adrenocortical carcinomas, adrenocortical adenomas, and pheochromocytomas were 85.9%, 69.6% and 71.4%, respectively, whereas the insulin receptors were expressed in 90.6%, 100%, and 100%, respectively. The frequency of insulin immunoreactivity and insulin receptor expression among these tumors was much the same. The intensity of staining was examined in the adrenal tumors. The IGF-1, IGF-I receptors, and insulin receptors were strongly stained in the adrenocortical carcinomas more than in the adrenocortical adenomas (Tables 2 and 3). In addition, the adrenocortical adenomas with Cushing's syndrome and pheochromocytomas showed a stronger intensity for insulin receptors than did the adrenocortical adenomas with Conn's syndrome (Table 4), although the intensity of IGF-I, IGF-I receptors, and insulin immunostaining apparently was not related to function of the tumor tissues (data not shown). To determine whether or not the receptor expression correlated with the immunoreactivity level, data were an-

alyzed by Kendall's tau. No relation was found in the adrenocortical adenomas and pheochromocytomas (data not shown). Conversely, in the adrenocortical carcinomas, the receptors for IGF-I and insulin significantly correlated with the intensity of IGF-I and insulin immunostaining, respectively, the relationship being positive (IGF-I: Kendall's tau = 0.403, P = 0.0023, insulin: Kendall's tau = 0.347, P = 0.0080) (Table 5). There was no significant correlation between IGF-I receptors and insulin receptors in the adrenocortical carcinomas (Table 6). The relationship between the intensity of IGF-I receptor staining and the histologic grading of adrenocortical carcinomas was negative (Kendall's tau = -0.447, P = 0.004) (Table 7). There was no significant relationship between the histologic grading of the adrenocortical carcinomas and the intensity of staining for IGF-1, insulin, or insulin receptors (data not shown).

Discussion We confirmed the presence of IGF-I and insulin immunoreactivities and receptor expressions in human adrenal tumors, including adrenocortical carcinomas, adrenocor-

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I

Table 2. Staining Intensity of IGF-I and IGF-I Receptors in Adrenal Tumors

Table 4. Immunostaining Intensity of Insulin Receptors in Functioning Adrenocortical Adenomas and Pheocbromocytomas

Staining intensity of IGF-I Adrenocortical carcinoma Adrenocortical adenoma* Pheochromocytoma x2

=

2

1

0 6

12

25

21

12 3

6

4 2

1 1

1

Staining intensity 1 2 3 0 6 5 1 2

3

0 0 0 0

Cushing's syndrome* Conn's syndrome*t Pheochromocytomat

3 6 0 4

X2= 11.919 (Cushing's syndrome vs. Conn's syndrome); P < 0.01. t X2 = 8.1820 (Pheochromocytoma vs. Conn's syndrome); P < 0.05. *

23.225 (carcinomas vs. adenomas); P < 0.01.

Staining intensity of IGF-I receptors

Adrenocortical carcinoma Adrenocortical adenomat Pheochromocytoma

0

1

2

4

20

27

13

1

13

7

2

2

t x2 = 8.786 (carcinomas vts.

3

31

1

well as in the cell membrane. Receptor-mediated endocytosis has been described for various ligands, includas

adenomas); P < 0.05.

ing IGF-I and insulin.9'37-39 The pathways that the receptorligand complexes follow inside a cell can be divided into four major classes,39 and all classes of receptor show the same initial step; the receptor-ligand complexes after binding at the cell surface internalize through coated pits and vesicles,

and ultimately enter acidic endosomal

partments.39 Hence, immunostaining in the cytoplasm is considered to identify the internalized ligand-binding retical adenomas, and phec ceptor proteins or degraded receptor proteins. This pheInotheiposi ibution of immunostaining was control tissues, the distr )chrtiomocytoma nomenon may be noted especially in the use of paraffin used only formalin-fixed the same as reported.133 X35We used only formalin-fixed etos and paraffin-embedded b-.issues. In the initial experiment, initialexperIn the non-neoplastic adrenal glands, there was a similar paraffin treatment disclos ed lesser sensitivity of the imdistribution of immunoreactivity and receptors between in the fresh fromunohistochemical assaMy' than IGF-I and insulin, although there were different intensities zen sections. However tl he ratio of positive cells in both of staining in each zone of the adrenal gland. Both IGF-I tissue preparations was similar. Moreover there was no and insulin are involved in the regulation of cell growth evidence of positive stair and steroidogenesis in the adrenal cortex, in the presence of immunogen peptide was preincubated with ar i or absence of angiotensin 11 and adrenocorticotropic horor when an irrelevant imr nunogloblin applied. mone (ACTH).16'17 In the adrenal medulla, both peptides Immunostaining for bioth IGF-I and insulin receptors also can increase catecholamine secretion from chromaffin was localized in the cytopl asm, with fine granular pattern, cells in response to high K+, with a different magnitude.' The existence of distinct receptors for IGF-I and insulin in the adrenal glands has been well documented.16'17'19'20 Table 3. Staining Intens ity of Insulin and Insulin .25'34

a

was seen

uingiwhen threoverpteri wanbod excess

serum was

a

Receptors in Adrenal Tumors

Staining intensity of insulin Adrenocortical carcinoma Adrenocortical adenoma

Pheochromocytoma

0

1

2

3

9

19

19

17

7 2

7 1

7 1

2 3

No statistical significance.

Table 5. Relationship Between Immunoreactivity and Receptor Expression in Adrenocortical Carcinomas IGF-I immunoreactivity 1 2 0 3 0 2 1 1 0 1 7 IGF-I 0 4 9 2 3 Receptors 3 13 8 1 2 1 3 9 Kendall's tau = 0.403; P = 0.0023.

Staining intensity of insulin receptors

0

1

2

3

Adrenocortical carcinoma Adrenocortical

6

5

28

25

adenoma* Pheochromocytoma

0 0

8

9

6

1

2

4

*2

=

11.277 (carcinomas vs. adenomas); P < 0.05.

Insulin immunoreactivity

0 Insulin Receptors

0 3 0 3 3

1 2 3 = Kendall's tau 0.347; P = 0.008.

1

2

2 4 9 4

1 1

8 9

3 0 0 8 9

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Of the adrenocortical carcinomas investigated, 90.6% showed a strong positivity for IGF-I, and the frequency was significantly higher than that seen in the adrenocortical adenomas and pheochromocytomas. The strong intensity of IGF-I receptors also was noted in adrenocortical carcinomas, although there was no difference in the frequency among the adrenal tumors examined. We found no significant relationship between immunoreactivity or receptor expression of IGF-I and function of the tumor tissues. Gourmelen et aW4I reported that serum concentrations of IGF-I for patients with Cushing's syndrome did not differ from normal values and did not reflect the severity of the hypercortisolism. Insulinlike growth factor I was seen to act as the growth factor for pheochromocytoma cells.2223 Furthermore, the intensity of IGF-I immunoreactivity was positively correlated with that of IGF-I receptors. Adrenocortical carcinomas seem to have characteristics of fetal adrenal glands,3 which express twice the concentration of IGF-I messenger ribonucleic acid (mRNA) than does the liver.42 Taken in conjunction with the observations that human neoplastic cells produce or release IGF-1-like peptides and express a high level of IGF-I receptors for proliferation of tumor cells,2'5'6'11-13 the possibility that IGF-I in particular is associated with growth of adrenocortical carcinomas, possibly through an autocrine mechanism, would have to be considered. Fitzpatrick et al43 reported that the expression of growth factor receptors significantly correlated with the histologic grading. With regard to assessments of histologic grading of adrenocortical carcinomas, we found negative correlation with the intensity of IGF-I receptor staining, thereby indicating that IGF-I receptors in the well-differentiated type were expressed at higher levels than in the poorly differentiated type. This finding supports the concept that IGFor its receptor is related to the function of differentiation of cells and tissues as well as to growth.44 We found no significantly different frequency of insulin immunoreactivity and insulin receptor expression between benign and malignant tumors of the adrenal glands. Eightyeight of 94 adrenal tumors examined expressed insulin receptors with different intensities, and only six of the adrenocortical carcinomas were devoid of positive cells. However the intensity of insulin receptor staining in the Table 6. Relationship Between IGF-I Receptors and Insulin Receptors in Adrenocortical Carcinomas

0 Insulin

0 1 1 2 0

1 2 3 Kendall's tau = 0.210; P = 0.1483.

Receptors

IGF-I receptors 1 2 1 4 1 1 11 11 7 11

3 0 2 4 7

Table 7. Relationship Between Histologic Grading and Staining Intensity of IGF-I Receptors in Adrenocortical Carcinomas

Staining intensity of IGF-I receptor 0

Gl G2 G3

1 2 1

1

5 10 5 Kendall's tau = -0.447; P = 0.004.

2

3

9 12 6

1 9 3

adrenocortical carcinomas was stronger than that in the adrenocortical adenomas. Like IGF-I receptors, the intensity between immunoreactivity and receptor expression of insulin showed a positive correlation in the adrenocortical carcinomas. Hence insulin also may play a role in growth of adrenocortical carcinomas, although insulin may be much less potent than IGF-I for the growth. We obtained no clear evidence of the coexpression of IGF-I and insulin receptors in the same carcinoma cells. However somatomedin C, which is considered to be identical to IGF-1, possesses a structural and functional homology with insulin.7'8 Receptors for IGF-I and insulin also share similar features with two a-subunits and two (3-subunits linked by disulfide bridges.9'10 Insulinlike growth factor and insulin each bind to its own receptor and, to a lesser extent, to the other receptor,9'10 and the mitogenic effect of insulin is considered to be mediated, in part, through the IGF-I receptors.45 Therefore, IGF-I and insulin may affect the adrenocortical carcinomas by receptor interaction, as well as through their own receptors. Immunostaining for the insulin receptors disclosed a stronger intensity in the adrenocortical adenomas with Cushing's syndrome and pheochromocytomas than in the adrenocortical adenomas with Conn's syndrome. A high concentration of insulin was noted to enhance independently the activities of steroid hydroxylase, such as 3fl-hydroxysteroid dehydrogenase/isomerase, and 21and 1 1,3-hydroxylase.1646 It is not clear whether the concentration of insulin is elevated in patients with Cushing's syndrome and in those with a pheochomocytoma. However the enhancement of insulin receptor expression does indicate that insulin, acting on its receptor, may play an important role in the regulation of glucocorticoid and catecholamine biosynthesis in the adrenocortical adenomas with Cushing's syndrome and pheochromocytomas. Tsugawa et a121 reported that insulin directly increased catecholamine secretion from human pheochromocytoma cells, the results of which differed from findings in normal bovine chromaffin cells.40 Whether insulin stimulates steroidogenesis in the adrenocortical adenomas with Cushing's syndrome is the subject of ongoing study. In conclusion, the immunoreactivity and receptor expression of both IGF-I and insulin were evidenced in 94

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adrenal tumors. The IGF-I and insulin probably act as autocrine growth factor in the adrenocortical carcinomas. In addition, IGF-I may also play a role in the function of differentiation of the adrenocortical carcinoma tissues. The possibility that the expression of insulin receptors may be associated with steroid-catecholamine biosynthesis in benign adrenal tumors is worthy of consideration. The expression of growth factors in neoplastic tissues may be related to the prognosis of patients.47 8We were not able to determine whether the growth factors and their receptors can serve as a prognostic parameter, because 96.9% of the adrenocortical carcinomas had already metastasized to other organs and because 78.3% of the patients died within 1 year of the onset of symptoms.

Acknowledgments The authors thank M. Ohara for critical comments, M. Inomata, Y. Yamashita, and S. Nakanose for technical assistance, and T. Hayashida for photographic services. They also thank Dr. M. Nakano, Department of Surgery, Maizuru Municipal Hospital, Kyoto, Japan, for help with collection of the tissue samples and the histological grading of the adrenocortical carcinomas.

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