Immunohistochemical localization of neuron-specific enolase in gastroenteropancreatic neuroendocrine...

lmmunohistochemical Localization of Neuron-Specific Enolase in Gastroenteropancreatic Neuroendocrine Tumors

Correlation With Tissue and Serum Levels of Neuron-Specific Enolase

SHANNON SIMPSON, MD,* AARON I. VINIK, MD,t PAUL J. MARANGOS, P H D ~ AND RICARDO V. LLOYD, MD, PHD'

Neuron-specific enolase (NSE) was studied by immunohistochemistry and radioimmunoassay in ten gastroenteropancreatic (GEP) neuroendocrine tumors. Tissue and serum levels of NSE from the same patients were also analyzed. In all cases, NSE was localized by immunohistochemistry as diffuse cytoplasmic staining to neuroendocrine cells. Tissue levels of NSE were elevated in eight of ten cases while the serum level of NSE was elevated only in one patient with a metastatic gastrinoma to the liver. Examination of the distribution of NSE in a wide range of tumors (n = 55) showed that it is relatively specific for neurons and neuroendocrine tumors. Eight of ten tumors analyzed immunohistochemically for nine polypeptide hormones contained more than one hormone. Two of ten tumors with only one hormone were positive for NSE. The clinical syndrome in all cases was related to only one hormone. These results indicate that the immunohistochemical demonstration of NSE is a good general marker for the neuroendocrine system. While tissue levels of NSE in GEP neuroendocrine tumors are generally elevated, the serum levels of NSE may only be markedly elevated with extensive metastatic disease.

Cancer 54:1364-1369, 1984.

EUROENDOCRINE CELLS and tumors of the gastroen- N teropancreatic (GEP) system have been shown to produce many different hormonally active peptides and amines by immunohistochemical studies. Neuron-specific enolase (NSE) has recently been described as a general marker of neuroendocrine cells and turn or^.^.^ Initially found in neuronal cells, NSE is also present in peripheral nerves and neuroendocrine tumors including neuroblas- tomas, medullary thyroid carcinomas, melanomas, and pheochromocytomas.3-7 Therefore, NSE can serve as a common marker for neuroendocrine tissues. NSE is also present in metastatic neuroendocrine tumors and has been identified in clinically silent tumors as well as in tumors that secrete one or multiple hormones.'

Serum levels of NSE have been shown to be elevated in pulmonary neuroendocrine tumors, and this is more marked in patients with metastatic disease.'.'' Although several reports have examined tissue and serum levels of NSE, determination of NSE by immunohistochemistry

From the *Department of Pathology, University of Michigan, Ann Arbor, Michigan, ?Department of Internal Medicine (Division of En- docrinology and Metabolism Research Unit), Surgery and Radiology, University of Michigan, and $Laboratory of Clinical Science, National Institutes of Mental Health, Bethesda, Maryland.

The authors thank Dr. R. Chance for the antiserum to pancreatic polypeptide; Pam Chlebek for typing the manuscript, and Alexandra Parma for excellent technical assistance.

Accepted for publication July 18, 1983.

and correlation with tissue and serum levels in patients with pancreatic endocrine tumors has not been previously reported.

In this study ten gastrointestinal (GI) neuroendocrine tumors were examined immunohistochemically for specific polypeptide hormones and NSE. Serum and tissue levels of NSE were determined and correlated with the clinical findings.

Materials and Methods

Gastroenteropancreatic neuroendocrine tumors from ten patients were examined. Clinical details of the ten patients studied are summarized in Table I. Serum sam- ples for NSE were obtained before surgery, and tumor tissues were obtained at the time of operation in ten patients with neuroendocrine tumors. Formalin-fixed, par- affin-embedded tissues were cut at 4 pm and stained by immunoperoxidase using the Avidin-Biotin Complex Method (ABC) with an unlabeled primary antibody, bio- tinylated secondary antibody, and a preformed avidin:biotin horseradish peroxidase complex (Vector Labs, Burlingame, CA).' ' , I 2 Tissues were treated with diami- nobenzidine and counterstained with hematoxylin. Sources of antisera and dilutions used were as follows: insulin 1/1000 (Dr. Besch, Indiana Univ.), glucagon 1/ 10 (Calbiochem, La Jolla, CA), human pancreatic poly-

1364

No. 7 NEURON-SPECIFIC ENOLASE IN GI TUMORS - Simpson et al. 1365

TABLE 1. Clinical and Pathologic Data of Patients With Gastrointestinal Tumors

Elevated Tumor type Tumor size (cm)

Case Age no. (yr) Sex Signs and symptoms serum level

I 57 M Hypoglycemic episodes Insulin Pancreatic insulinoma 1 .0

2

3

4

5

6

I

8

9

10

23

52

33

28

47

56

33

52

63

F

M

M

M

F

M

F

F

F

for 1 yr

for 10 yr

hypercalcemia

Hypoglycemic episodes

MEN I, gastric ulcer,

Hypoglycemic episodes for 4 yr

Hypoglycemic episodes for 1 yr

Perforating duodenal- jejunal ulcer consis- tent with Z-E syndrome ulcer symptom- atology for 5 yr

Gastritis, severe diarrhea

Abdominal pain, diarrhea for 3 yr

Epigastric pain flushing, weakness diarrhea for under 1 yr

Watery diarrhea, weight loss for 9 mo

Insulin, SRIF

PP, insulin

Insulin

Insulin

Gastrin

-

Gastrin

Gastrin

VIP. PP

Pancreatic insulinoma and somatostatinoma

Pancreatic adenocarcinoma, multiple adenomas, insulinoma and PPoma, nesidioblastosis

Pancreatic insulinoma

Pancreatic insulinoma

Peripancreatic gastrinoma

Pancreatic nesidioblastosis, metastatic apudoma to liver

Peripancreatic gastrinoma

Gastric carcinoid

Pancreatic ppoma

2.0

Largest adenoma 0.8

2.6 X 2.4 X 1.4

1.5 X 0.7 X 0.6

2.0 X 1.5 X 0.65

Multiple 0.5 to 1.0, metastatic hepatic nodules

4.0 X 1.5 X 1.5

Three polyps, largest 1.5

5.3

Case 7 had metastatic gastrinoma in the liver. SRIF somatotropin-release inhibition factor; MEN: multiple endocrine

peptide (PP) 1/2000 (Dr. R. Chance, Eli Lilly), somatostatin 1/ 100 (Immunonuclear, Stillwater, MN), vasoactive intestinal polypeptide (VIP) 1 /2 (Immulok, Car- penteria, CA) adrenocorticotropic hormone (ACTH) l / 1000 (Dako, CA), calcitonin 1/200 (Immunonuclear, MN), serotonin 1/200 (Immunonuclear, MN), gastrin 1/ 10 (Calbiochem), NSE 1/500 (Dr. P. Marango, National Institutes of Mental Health, MD). The antisera against glucagon, gastrin, and VIP were previously diluted by the distributors. Serial sections of each tumor were incubated with the antisera. As positive controls, we used normal pancreas for insulin, glucagon, somatostatin, and PP, and gastric antrum for gastrin. The primary antisera were omitted for negative controls. In addition, the specificity of each antiserum was examined by specific absorption with appropriate antigens. The concentrations of antigen used to absorb the antisera ranged from 10 to 100 ng/ ml of purified antigens.

Serum and tissue levels of NSE were assayed by using previously published methods. ' Normal human serum contained less than at 5 ng/ml of NSE. Normal pancreatic tissues contained less than 50 ng/mg tissue protein.

Nesidioblastosis was defined as a diffuse or disseminated proliferation of islet cells closely associated with endocrine ducts and ductule~.'~ Endocrine hyperplasia was defined as the presence of nesidioblastosis or enlarged and irregular

neoplase; P P pancreatic polypeptide; Z-E: Zollinger-Ellison; VIP: vasoactive intestinal polypeptide; PPoma: pancreatic polypeptidoma.

islets with and without a normal distribution of cell types.I4

Results

All normal pancreatic islets (Fig. 1) showed diffuse staining for NSE as did areas of nesidioblastosis. In all

FIG. 1. Immunohistochemical staining of a normal pancreatic islet for NSE. There is diffuse staining of all cells in the islet. (Immunoper- oxidase, original magnification X330).

1366 CANCER October 1 1984 VOl. 54

FIGS. 2A AND 2B. (A, top) Immunohistochemical staining of an insulinoma for NSE. The tumor cells show a diffuse cytoplasmic staining for NSE. (Immunoperoxidase, original magnification X330). (B, bottom) Immunohistochemical staining of the same insulinoma shows the juxtanuclear localization of insulin, which is distinct from the diffuse cytoplasmic localization of NSE. (Immunoperoxidase, original magnification X330).

ten neuroendocrine tumors, NSE was identified immunohistochemically (Fig. 2A). The staining vaned from weak to intense and was present diffusely in the cytoplasm. The majority of the neuroendocrine tumors had elevated NSE tissue levels. The degree of elevation in general correlated with the intensity of the immunocytochemical staining. One case with an elevated serum level of NSE was also the only case with metastatic disease (Table 2). This tumor also had the lowest level of NSE. The specificity of NSE for neuroendocrine tissue was also demonstrated (Table 3). Normal neurons and adrenal medullary cells also contained NSE. Other tumors that stained positive for NSE included carcinoids, melanomas, and medullary thyroid carcinomas (Table 3) .

The presence of multiple polypeptide hormones in several tumors was also demonstrated (Table 4). The presence

of more than one hormone was found in eight of the ten tumors by immunohistochemical localization. In two of the eight cases (cases 2 and 3), there was elevation of two hormones in the serum. However, in all instances the clinical syndrome could be attributed to overproduction of one hormone. The majority of the tumors showed diffuse staining for polypeptide hormones. In one case, insulin was preferentially localized in small globules in juxtanuclear areas (Fig. 2B).

Adrenocorticotropic hormone (ACTH) was not identified in any of the tumors and calcitonin was localized in one tumor.

Discussion

Gastroenteropancreatic endocrine tissue and tumors have many characteristics in common with amine precursor uptake and decarboxylation (APUD) tissues, although these cells are not of neural crest rigi in.'^,'^ The APUD system incorporates cells with common cyto- chemical characteristics related to the synthesis of poly- peptides and amines and includes endocrine cells of the pancreas and other tissues.16 These tissues and tumors with APUD characteristics form part of the diffuse neuroendocrine system (DNES).I7 It is becoming increasingly clear that NSE is a common marker for APUD cell^^,^ and that its quantitation and immunohistochemical vi- sualization can be of value in characterizing cells and tumors of the DNES.

Our results indicate that NSE is a good immunohistochemical marker for GI neuroendocrine tumors and normal islet cells. Neuron-specific enolase is found in most neuroendocrine cells and in neuron^.^,^^^ Enolase is a glycolytic dimeric cytoplasmic enzyme (2-phospho-D glycerate hydrolase). It consists of three subunits: a, P, y arranged into five possible dimeric isoenzymes. The a homodimer is the most common form in adult tissue. The P homodimer is present mainly in muscle. The yy NSE is found in neurons and neuroendocrine tissue. The origin and distribution of the two hybrid forms (aP, rP) has not been completely elucidated. 18-20 Neuron-specific enolase appears to be specific for neuroendocrine and nervous tissue.'^'' Of 65 tumors examined, NSE was found in 42 or 45 endocrine tumors. One of two papillary thyroid carcinomas was focally positive and 0 of 20 nonendocrine tumors were positive for NSE.

Tissue levels of NSE were elevated in all but two of ten GI endocrine tumors (Cases 3 and 7). Case 3 also contained very large areas of pancreatic adenocarcinoma. The adenocarcinoma cells were negative for NSE. The presence of the adenocarcinoma in the tissue extracts may have resulted in the apparently normal tissue levels of NSE. Similarly, Case 7 contained some areas of normal liver parenchyma, which may account for the normal tissue level of NSE.

No. 7 NEURON-SPECIRC ENOLASE IN GI TUMORS Simpson et al. 1367

TABLE 2. lmmunohistochemical Localization of Neuron-Specific Enolase in Neuroendoendocnne Tumors and Associated Serum Levels and Tissue Levels

NSES

Serum levels Tissue levels Tumor type* Location NSET (nglml) (ng/mg protein)

~~~ ~

No. I , insulinoma No. 2, insulinoma, somatastatinoma No. 3, PPoma, insulinoma No. 4, insulinoma No. 5, insulinoma No. 6, gastrinoma No. 7, gastnnomas No. 8, gastrinoma No. 9, carcinoid No. 10, PPoma, vipoma

Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Liver, nesidoblastosis in pancreas Peripancreatic tissue Stomach Pancreas

+ + + + + + + + + +

4.3 5.3 4.5 5.3 5.3 5.2

14.7 7.2 8.1 3.9

182.9 267.1

26.7 163.5 378.6 91.7 19.6

123 1 I7 527.7

* Tumor type determined by appropriate clinical testing, and serum levels, and transvenous hepatic sampling of levels of gastrointestinal and pancreatic hormones.

t lmmunohistochemical localization of NSE.

One patient (Case 7) had slightly elevated serum levels of NSE and serum gastrin. This patient was the only one in whom there was metastatic disease. The primary tumor was not identified in this patient. A distal pancreatectomy, gastrectomy, and splenectomy were performed and nesidioblastosis was noted in the pancreas but no endocrine tumor was found. Other studies have found that the serum level of NSE correlates with the tumor burden."." A recent study of small cell lung carcinoma found that the degree of and percentage of patients with elevated serum NSE correlated with the extent of the disease." Serum NSE levels were elevated in 39% of patients with limited stage disease and in 87% of patients with extensive disease.' All patients with small cell lung carcinoma who had metastases at three or more sites had an elevation of serum NSE.' Prinz and Marangos" found that 9 of 2 I patients with a wide range of neuroendocrine tumors, including 9 of the GI tract, had elevated serum NSE levels. Four other patients had levels slightly higher than control values but not in an unequivocally abnormal range." Nine of 15 cases with metastatic disease had elevated NSE levels, which correlated with the extent of tissue involved. Five of the patients with normal and borderline levels of serum NSE had limited metastases. The remaining patient with a gastrinoma and hepatic metastases had borderline elevation of serum NSE, which was not explained." In our cases, elevation of serum NSE did not correlate with tumor size. However, the patient with the longest tumor (Case lo), which was benign, had the highest tissue level and the lowest serum level of NSE. In contrast, Patient 7 had multiple small metastatic nodules in the liver. In this patient the tumor levels of NSE were low while the serum levels were raised. Thus serum levels of NSE may be a very useful clinical adjunct to evaluating the presence of malignancy at the time of di-

$ Normals: serum NSE < 5 ng/mI, pancreatic tissue NSE (50 ng/

NSE neuron-specific endolase; PPoma: pancreatic polypeptidoma. mg tissue protein.

agnosis. A raised serum level in a patient with a neuroendocrine tumor suggests metastatic disease.

Neuron-specific enolase appears to be a tissue-specific marker for neuroendocrine tumors, a finding that is con- firmed in this article. Immunohistochemical staining for NSE is an excellent general marker for neuroendocrine neoplasms, especially when the battery of antisera nec- essary for identification of specific hormonal products and electron microscopic studies are not available. NSE

TABLE 3. Distribution of Neuron-Specific Enolase in Endocrine and Other Tumors

Tumor Result*

Melanoma Paraganglioma Pheochromocytomas Carcinoid Nevus Neuroblastoma Neuroendocrine carcinoma of skin Thyroid

Papillary carcinoma Medullary thyroid carcinoma

Pituitary adenoma Parathyroid adenoma Breast adenocarcinoma Lung

Squamous cell Adenocarcinoma

Colon adenocarcinoma Kidney

Wilms' Tumor Renal cell adenocarcinoma

Lymphoma Malignant schwanoma Malignant fibrous histiocytes Prostate adenocarcinoma Leiom yosarcoma

* No. of positive cases per total number of case examined. Focal staining only.

1368 CANCER October 1 1984 VOl. 54

TABLE 4. Immunohistochemical Localization Polypeptide Hormones

Tumor type Gastrin SRIF Serotonin PP Glucagon Insulin VIP

- + - No. I , insulinoma - - - No. 2. insulinoma,

somatostatinoma + + +, f +, f No. 3. PPoma,

insulinoma - No. 4, insulinoma - No. 5 , insulinoma - -

No. 6, gastrinoma + No. 7, gastrinoma + No. 8, gastrinoma + No. 9, carcinoid - - - -

No. 10, PPoma

+ - +, f -

- + + + + +

+ +

- + - - -

- - - - +, f

- - - + + +

- - - - - -

- - - - -

- - + + - - + - - - vipoma

ACTH: all tumors negative: calcitonin: only Case 10 was positive for

SRIF: somatotropin-release inhibition factor: PP: pancreatic poly- calcitonin.

peptide: VIP: vasoactive intestinal polypeptide; PPoma: papilloma; ACTH adrenocorticotropic hormone; f: focal; +: positive staining; -: negative staining.

is also a more general marker for neuroendocrine tissue than agryrophilic or argentaffin staining, since not all neuroendocrine tissues are positive by silver staining.

As many as 50% of GEP neuroendocrine tumors produce more than one hormone.' Three types of GI neuroendocrine tumors have been described (1) a single tumor with multiple cell types; (2) multiple tumors com- posed of one cell type; and (3) multiple tumors, one or more consisting of multiple cell types.' Eight of our cases contained more than one hormone by immunohistochemistry. Two cases had multiple tumors, one case producing the same hormones (insulin and PP) in each tumor, and the other case was a gastric carcinoid with multiple tumor nodules each of which was producing serotonin. Patients with multihormonal tumors usually have only one dominant clinical syndrome. In our cases of multihormonal tumors, the one predominant hormone de- tected immunohistochemically generally correlated with serum hormone levels and with the clinical signs and symptoms. The hormones produced by the primary tumor may be different from these in the metastatic foci. A recent report indicated that while a pancreatic tumor was producing insulin, gastrin, and glucagon, hepatic metastases from the same patient contained only glucagon and gastrin.2' PP is the most commonly seen hormone in multihormonal tumors. In our study, PP was present in six tumors, but in only one case (Case 10) was it part of the diarrhea syndrome. In this patient, VIP and calcitonin were also found in the tumor. Calcitonin has also been reported in some endocrine turn or^.^^-^^ In our case with diarrhea in which calcitonin was found in the tumor (Case lo), the serum calcium was normal. A recent study found both ACTH and calcitonin in tumor extracts of 12 of 42 a p u d o m a ~ . ~ ~

Elevated serum levels of PP have been proposed as a marker for the presence of pancreatic apudomas in the multiple endocrine neoplasm (MEN) I syndrome.25 We

have found this to be useful in certain pancreatic endocrine tumors.26 Two of our cases had an elevated serum PP (Cases 3 and 10) and only one (Case 3) was a com- ponent of the MEN syndrome (Tables 3 and 4). Our case of MEN I syndrome also had a pancreatic adenocarcinoma. A recent report has described a specific pattern of hyperplastic PP and insulin cells in close association with invading carcinomatous ducts in pancreatic adenocar- ~ i n o m a . ~ ~ This was not noted in our case with adenocarcinoma (Case 3). The presence of endocrine tumors associated with a pancreatic adenocarcinoma is note- worthy as in our Case 3, since the increased incidence of multiple primary carcinomas and endocrine tumors in other organs with pancreatic adenocarcinoma has been recently reported.28

Islet cell hyperplasia has been divided by Larsson into types I and II.29 Type 1 consists of prominent nesidioblastosis and hyperplasia of all islet cell types, especially insulin and glucagon cells. Type I hyperplasia is often seen with the Zollinger-Ellison (Z-E) syndrome and is believed to be caused by stimulation from increased gastrin. Type I1 hyperplasia consists of PP cell proliferation and is often seen with endocrine pancreatic tumors other than in patients with the Z-E syndrome and with pancreatic fibrosis.28 In contrast to Larson's findings, only one (Case 7) of our four cases (Cases 6-9) with increased serum gastrin had type I hyperplasia. Our only case with typical Z-E syndrome did not have type I hyperplasia. However, several of our cases did show proliferation of PP cells within the tumor.

In conclusion, our findings demonstrate that the immunohistochemical demonstration of NSE is a good general marker for tumors of the neuroendocrine system and can be useful in the classification of tumors as either endocrine or nonendocrine. Serum NSE may not be a sensitive marker for pancreatic endocrine tumors, except for cases with metastatic disease.

No. 7 N’URON-SPECIFIC ENOLASE IN GI TUMORS - Sirnpson et al. 1369

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