Localization and Peptide Content Endocrine Pancreatic Tumors
of
HANS MARTENSSON, M.D., PH.D.,* GERHARD BOTTCHER, M.D.,t FRANK SUNDLER, M.D., PH.D.,t and ANDERS NOBIN, M.D., PH.D.*
Endocrine pancreatic tumors contain and frequently secret neurohormonal peptides. This phenomenon can be used as a diagnostic and classifying tool. This study analyzes 31 patients operated on because of an endocrine pancreatic tumor, including the diagnostic procedures and the localization methods. In 15 insulinoma cases only 6 patients had a positive arteriography, while all 11 selective pancreatic vein samplings were positive. The immunoreactivity showed that, besides insulin, most tumors also contained other peptides. Of four gastrinoma cases the arteriography was positive in three, but the selective vein sampling localized the tumor in all. The tumors's content of peptides showed mixed patterns. In the four glucagonomas, the arteriography was positive in all and the venous sampling performed in three of the cases also was positive. In five pancreatic polypeptide-containing tumors (PP-omas) the arteriography was positive in four and sampling performed in two was positive in both. In the PP-omas the peptide pattern showed that these tumors frequently contain several peptides. We used selective pancreatic vein sampling in 21 cases with positive result in all. In the cases in which arteriography was negative, the sampling results helped the surgeon to find the tumor. The peptide pattern in the tumors varied greatly and most tumors were multihormonal. E a NDOCRINE TUMORS OF the pancreas are rare, but
when they do occur the patients may suffer considerably before diagnosis is reached and the tumor is removed. In one important aspect endocrine tumors are different from other tumors arising in the pancreas. They produce neurohormonal peptides in varying degrees, which, in many cases, can be analyzed by radioimmunoassay. X This fact makes is possible to use neurohormonal peptides as tumor markers aiding in the diagnosis by measuring elevated fasting levels in the blood.2'3 The development of stimulation and suppression tests has increased the sensibility and diagnostic reliability even
more.4'5
From the Departments of Surgery* and Histology,t Lund University, Lund, Sweden
The true incidence of endocrine pancreatic tumors is difficult to estimate because many of these tumors are silent. However the distribution of various types of islet cell tumors is documented in several series.6'7 The most common tumor is insulinoma (50% to 60%), followed by 'nonfunctioning' tumors (15% to 25%), gastrinoma (18% to 20%), and various types (7%). Once the diagnosis is established, the question arises of where the tumor is localized within the pancreas and whether it is solitary or multiple. The noninvasive preoperative localization methods of choice are sonography and computed tomographic (CT) scanning. Sonography has been shown to be positive in 63% to 90% of cases, and CT scan is positive in 45% to 90% of cases, as compiled from several studies.8"1l Arteriography is an invasive examination with an accuracy rate of 40% to 90% and a false-positive rate of about 5%.8,9,11 The abnormal release of neurohormonal peptides from the tumors has enabled their localization by sampling blood from various branches of the portal or intestinal veins. This method, however, is highly invasive and the outcome depends on the skill of the investigator. The reported results are thus poor (60% positive) when the sampling is done in the main portal branches; they are much better, however (80% to 90% positive), when a superselective sampling is made.8 Since the early 1970s we have used the superselective sampling technique for the localization of different gastrointestinal endocrine tumors and their metastasized
areas.12 Some gastroenteropancreatic endocrine tumors behave
Address reprint requests to Hans Martensson, Department of Surgery, Helsingborg Hospital, S-251 87 Helsingborg, Sweden. Accepted for publication January 10, 1990.
more aggressively than others of the same size and extent of metastasis. Therefore to obtain prognostic information, analyses of growth pattern, peptide content, and patterns
607
608
MARTENSSON AND OTHERS
of DNA distribution in carcinoid tumors have been made, but without consistent results.'3 Hence the treatment strategy of gastroenteropancreatic endocrine tumors is still surgical intervention. To achieve radicality a preoperative mapping of the tumor extent is essential. A number of investigations have shown that endocrine pancreatic tumors frequently are multihormonal, i.e., contain several neurohormonal peptides. 14-16 Such tumors are composed of multiple endocrine cell types, some of
Ann. Surg. * November 1990
which are not seen in the normal adult pancreas. For several peptides there are reports also on elevated plasma levels in patients with pancreatic endocrine tumors.'7 It is also noteworthy that the metastasized areas may contain a different set of peptides than the primary tumor. In this series we report our experience with pancreatic vein catheterization with blood sampling to localize 31 pancreatic endocrine tumors treated at our department. Because it cannot be excluded that the peptide content
FIG. 1. Sections from two insulinomas immunostained for chromogranin A (upper left [a] and upper right [bi; 350X) and for insulin (lower left [cJ and lower right [d]; 250X and IOOX). Most of the cells display chromograninand insulinfluorescence of varying intensity. In [d] in-
tenselycinsulin-immuno-
*
~~~reactive cells
occur among the more weakly stained ones.
VOl. 212.- NO. S
ENDOCRINE PANCREATIC TUMORS
609
FIG. 2. Two insulinomas immunostained for insulin (left [a] and middle [b]; 175X). The immunoreactive cells display slightly different growth patterns. Some insulinomas also contained single scattered gastrin-immunoreactive cells (right [c]; 300X).
and pattern in the tumor may be related to the aggressiveness of the disease, we re-examined all tumor specimens available with respect to their peptide content and distribution as demonstrated by immunocytochemistry. Materials and Methods The records of 31 patients operated on because of a suspected pancreatic endocrine tumor were reviewed. The analysis encompassed clinical picture, patient age, time from start of symptoms to diagnosis, diagnostic procedures, localization methods, type of operation, tumor size and number, extent of metastasis, and survival. The pancreatic vein sampling was performed using techniques previously published from our hospital.12 Tumor tissue specimens available were re-examined using immunocytochemistry with respect to the content of neurohormonal peptides and serotonin. However none of the three islet cell hyperplasias was available for reexamination. Most pancreatic specimens, or metastasized areas, were obtained at surgery and fixed on either 4% phosphate buffer (pH 7.3; 1.5 mol/L [molar]) formaldehyde or on Bouin's solution (4% formaldehyde, 1% picric acid, 0.5% acetic acid) for 18 hours. The specimens were dehydrated through a graded series of alcohol, embedded in paraffin, and sectioned at ± 5-,tm thickness. The hydrated solutions were processed for fluorescence or peroxidase indirect immunocytochemistry'8 using primary antisera to insulin, glucagon, somatostatin, pan-
creatic polypeptide (PP), gastrin, Pro-ACTH, calcitonin (CT), calcitonin gene related peptide (CGRP), PYY, Galanin, or serotonin. Results The tumors comprise 15 insulinomas, of which 3 were islet cell hyperplasias, 5 gastrinomas, 5 pancreatic polypeptide-containing tumors (PP-omas), 4 glucagonomas, and 2 unclassified tumors. The tumor classification is based on the clinical symptoms, except for PP-omas, in which the main peptide released from and/or contained in the tumor formed the basis. From one of the two unclassified tumors, specimens were not available for re-examination and the other showed no immunocytochemical
reaction. Insulinomas Of the 15 patients, there were 11 women and 4 men, with a mean age of 57 years (median, 60 years; range, 32 to 83 years). The main symptoms were disturbances in consciousness in 14 patients, fatigue in 7 patients, and weight gain in 2 patients. Most patients were diagnosed within 1 year. In two patients, however, the time from onset of symptoms to diagnosis was more than 3 years. Fasting test with monitoring of insulin-glucose ratio was performed in 12 patients, and all were positive within 24 hours (Table 1). Arteriography was undertaken in 15 patients but was positive in only six patients (Table 1). A selective pancreatic vein sampling was performed in 11 patients, which resulted in correct localization of the tu-
610
Ann. Surg. * November 1990
MARTENSSON AND OTHERS
II
11
FIG. 3. Two glucagon-containing tumors (left [a]; 175X, and middle [b]; 200X). Note the different growth pattern and the different arrangement of glucagon cells in the two tumors. At the right, [c]; 200X, a section from the tumor shown in [b] was immunostained for PP. PP-immunofluorescent cells occur scattered in the tumor.
mor in all cases (Table 1). The mean ratio ofpeptide level between a tumor-draining vein and a large vein was 18.8
to
1.
Surgery consisted of enucleation in case, tail resection in 10 cases, and Whipple's operation in case. The tumor was solitary in all the cases and the size of each tumor is listed in Table 1. All operations were considered radical, and no patient has died from an insulinoma. The results from immunocytochemical analysis are schematically shown in Table 2A. All tumors were im-
munoreactive for insulin, 2 tumors had more peptide, 2 tumors had 2 more peptides, 2 tumors had 3 more peptides, and 1 tumor had 5 more peptides (Figs. and 2). Only one tumor contained immunocytochemically demonstrable serotonin (Table 2 A).
Gastrinomas Patients with grastrinomas included two women and three men. Their mean age was 44 years (median, 42 years;
TABLE 1. Insulinomas. Some Clinical Characteristics and the Result of Localization Procedures
Patient
Pancreatic Vein Sampling Age
No.
Sex
(years)
I 2 3 4 5 6 7 8 9 10 11 12 13 14 15
F F F F F F F F F F M M M M F
67 74
82 68 52 41 40 37 32 61 60 62 72 55 62
Tumor Size (mm) 10 40 10 20 20 ? 8
Hyperplasia Hyperplasia 15 30
Hyperplasia 30 12 ?
Arteriography
CT Scan
Sonography
Fasting
+
ND
+
+
ND ND
+ + + + + + +
ND ND ND ND ND ND
+ +
+ +
+
ND ND ND
-
F, female; M, male; ND, not done; + positive localization;
-
+
ND ND ND ND ND ND ND
negative localization.
Result
Tumor Vein
Other Vein
179 ND
+
6200,uU/mL
ND
ND
+ + + + + + + +
308 7317 >400 >400 >400
+ +
ND
ND
ND
ND 170 ND
21 27 24 45 95 ND 8 ND
ND
ND
+
ND ND
+
+
+
+
ND
245 1130 704
131 145 170
Vol. 212 * No. 5
ENDOCRINE PANCREATIC TUMORS
611
TABLE 2. The Immunocytochemical Staining Pattern
A
Pro-ACTH
CT
PYY
CGRP
Galanin
Serotonin
-
-
+
-
-
-
-
-
-
_ _
-
-
-
-
_ _
+
_ _
_ _
+
-
-
-
-
+
-
-
-
ND
ND ND +
-
+
ND ND
ND
-
-
-
+
-
Insulin
Glucagon
Somatostatin
2 3 4
+ + + + + +
-
-
-
-
-
-
+
+
-
-
-
-
-
+
-
-
+
+
-
+
+
+ + +
-
+ +
+
13 14
-
-
-
-
-
-
-
-
-
-
I
-
-
-
+
-
-
-
+
-
-
2 3 4 5 3 4
+ +
+
-
+ +
-
+
_
+
-
+
-
-
-
-
-
+
+ -
ND -
ND -
_ _ ND ND
-
+ + -
_ _ ND ND
-
+ -
_ _ ND ND ND ND
+
-
5
6 7 10 11
B
C
Gastrin
Patient
-
1
+
2
-
-
4 5
+ -
_
+ -
-
+
-
+ +
D
PP
_ ND ND ND ND
ND
+
ND
ND
-
+
-
-
-
-
+
-
-
-
+
-
-
ND
ND
ND
ND
+
-
+
+
_
_
+
-
+
+ +
_
-
-
+
+
+
_
_ _
A, insulinomas; B, gastrinomas; C, glucagonomas; D, PP-omas; + positive immunoreactivity; - negative immunoreactivity; ND, not done. range, 34 to 54 years), and the main symptom was recurrent ulceration. Two patients had perforating ulcers, of which one was situated in the proximal jejunum and
the other in the proximal duodenum. Two patients had symptoms for more than 15 years, 1 patient for 3 years, and 2 patients for less than 1 year before diagnosis was established. Basal gastrin levels were increased in all patients (Table 3). A secretin provocation test was performed in three patients: all of them responding with excessive release of gastrin. Arteriography was performed in four patients, with a positive localization in three. Selective pancreatic vein sampling in all four patients localized the tumor in all (Table 3). The mean ratio of peptide level between a tumor-draining vein and a largeig vein was 4.4 to 1. In three patients a total gastrectomy was performed before the pancreatic surgery. A tail resection was performed in three patients; in one patient the tumor was enucleated, and in one patient a biopsy was performed.
One patient had a solitary tumor, three had multiple, and had a large mass in the pancreas. The diameter of the tumors ranged between 15 and 25 mm. One patient had no metastasis, two had lymph node metastasis, and two had liver metastasis. None of the patients died from the gastrinoma, and the two with liver metastasis are free from symptoms. Three of the patients were categorized as multiple endocrine neoplasia type I (MEN I). The immunocotychemical results are shown in Table 2B. One tumor contained no immunocytochemically demonstrable gastrin but five other peptides, and in another tumor only gastrin could be demonstrated. One tumor contained one more peptide and two tumors had two more peptides. None of the tumors contained serotoninimmunoreactive cells. one
Glucagonomas There were two women and two men aged 45, 63, 62, 69 years, respectively. The symptoms in all patients were
TABLE 3. Gastrinomas. Some Clinical Characteristics and the Result of Localization Procedures
Patient No.
Sex
Age (years)
1 2 3
M F M M F
52 38 42 54 34
4 5
Tumor Size (mm)
? 15 15 25 ?
Pancreatic Vein Sampling
Arteriography
CT Scan
Sonography ND ND ND ND ND
+
+
ND
ND
+ +
+
-
ND ND
F, female; M, male; ND, not done; + positive localization,
-
Secretin Test
Gastrin Result
ND
+
+
ND
ND
+ + +
negative localization.
+ +
Tumor Vein
Other Vein
ND >5000 776 2380
ND 500 424 1720
Basal Level
1100 pg/ml 587 470 8200
MARTENSSON AND OTHERS
612
Ann. Surg. * November 1990
TABLE 4. Glucanomas. Some Clinical Characteristics and the Result of Localization Procedures Patient No.
Sex
I
F M F M
2 3 4
Age (years)
63 62 45
69
Pancreatic Vein Sampling
Tumor Size (mm)
Arteriography
CT Scan
Sonography
Result
Tumor Vein
Other Vein
? 20 50 40
+ + + +
ND ND ND ND
ND ND ND -
ND + +
ND 24,000 pg/mL
ND 1540
+
550
10,000
Glucagon Basal Level
Normal 1500 87 1850
F, female; M, male; ND, not done; + positive localization; - negative localization.
diabetes and skin rash. Basal levels of circulating glucagon increased in three patients (Table 4). The time elapsing from the start of symptoms to diagnosis was 2, 4, 6, and 7 years. Arteriography was performed in all patients, and selective pancreatic vein sampling in three patients, and all were positive (Table 4). The mean ratio of peptide level between a tumor-draining vein and a large vein was 16.9 to 1. One patient had a portal occlusion that prevented portal vein catheterization. Surgery consisted of biopsy in 1, enukleation in 1, and tail resection in 2 patients. All tumors were solitary and measured 20 to 40 mm in diameter. Before operation none of the patients had signs of metastasis. At surgery one patient had a large mass in the pancreas, while the others had no signs of metastasis. However after operation liver metastasis developed in one patient and another patient has increased blood glucagon levels. Two glucagonoma tumors were available for re-examination (Fig. 3). One contained only glucagon and the other also contained somatostatin and serotonin (Table 2C). were
PP-omas Of the patients with PP-omas, there were four women and one man who were aged 55, 55, 59, 70, and 37 years, respectively. Two patients had no symptoms while three had diffuse abdominal pains. The tumors were discovered during investigation for pains or incidentally at surgery for other reasons. Basal plasma levels of PP were increased in 3 patients, normal in 1, and were not measured in 1
patient. Arteriography was performed in all patients and positive in four. Selective pancreatic vein sampling done in two of the patients and was positive in both (Table 5). The mean ratio of peptide level between a tumor-draining vein and a large vein was 4.3 to 1. Arteriography failed to produce positive results in the patients lacking increased levels of PP in blood. Two patients were operated on with the Whipple procedure, while from the others only biopsies were taken. The tumors were solitary, ranging in diameter size from 25 to 50 mm. Four patients had liver metastasis and one had lymph node metastasis. One patient died 4 years after diagnosis and three died within year. The only survivor (at 5 years) was the patient with lymph node metastasis. Four PP tumors were available for re-examination and the immunocytochemical results are shown in Table 2D. All tumors contained PP, had another 3 peptides, had 4, and had 5 more peptides. Only one tumor contained only PP. No serotonin was found in these tumors. was was
Discussion
Endocrine pancreatic tumors are rare, but when they do occur they may cause specific symptoms leading the physician to make an accurate diagnosis.'9 With modern techniques the relevant neurohormonal peptides may be identified under basal conditions and after stimulation or inhibition.'9 The combination of symptoms and peptide analysis has improved the accuracy of diagnosis. The treatment of a patient with pancreatic endocrine
TABLE 5. PP-omas. Some Clinical Characteristics and the Result of Localization Procedures
Patient No.
Sex
Age (years)
1 2 3 4 5
F F F M F
70 55 55 37 59
Pancreatic Vein Sampling
Tumor Size (mm)
Arteriography
CT Scan
Sonography
50
-
-
30 30 25 ?
+ + + +
+
+ +
ND
F, female; M, male; ND, not done, + positive localization;
+ + -
ND ND +
negative localization.
Result
Tumor Vein
Other Vein
PP Basal Level
+
51 pmol/L 299gg/mL
8 131 ND ND ND
Normal 212 ND 15OO pmol/L
+ ND ND ND
ND ND ND
30000pmol/L
Vol. 212 * No. 5
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ENDOCRINE PANCREATIC TUMORS
tumor has been, and still is, primarily surgery with the aim of radical excision. When this is not possible, an extensive reduction of tumor mass is desirable. Generally it is agreed that medical treatment with cytotoxic agents or interferon should be used when surgery is not possible or not radical.2s22 To make surgery optimal, preoperative localization of the tumors and possible metastasized areas is needed. We used superselective pancreatic vein catheterization with blood sampling in 21 cases. In all of these cases the result was positive and helped the surgeon to find the tumor. It is noteworthy that some of the insulinomas measured 12 mm or less in size, but still the sampling provided data of peptide secretion. This is even more interesting because the corresponding arteriography in these cases was negative. The advantage with the sampling technique is that it may detect small tumors where other methods fail. Only in one case was the sampling hard to interpret despite a tumor size of 5 cm: a PP-oma patient in whom the tumor contained immunoreactive PP, insulin, and glucagon, but none of the peptides displayed elevated levels in the plasma samples. This may be explained by the fact that none of these peptides were released. One possibility to overcome this may be the use of stimulation test during the sampling,5 but so far we have not used that. Therefore an uncertain or negative result of sampling may be caused by lack of neurohormonal peptide secretion or, alternatively, by the secretion of a defect peptide or a peptide fragment not picked up by the radioimmunoassay. In the 15 insulinoma cases it is interesting to note that nine arteriographies (60%) were negative while all samplings were positive. A small tumor size is not likely to be the cause of this because some of them were large and arteriography in other small tumors was positive. One possible explanation is that the vascularity of the insulinomas may be different when compared to other endocrine pancreatic tumors, thus explaining the difference in accuracy of the arteriography. Previously we calculated the arteriovenous difference in the sampling technique for localization of gastroenteropancreatic tumors.12 The interpretation of peptide values in the pancreatic vein sampling was without problems in all patients but one in this study, whereas the peptide levels expressed as the ratio between a tumor-draining vein and a larger pancreatic or portal vein were high in all the cases. The problem case is illustrative with absolute values almost normal, but the ratio being 6.5 to 1. Therefore we stopped using the technique of measuring the arteriovenous difference. The absolute value and/or the ratio between a tumor-draining vein and a large vein seems to give the same information and the patient is relieved from one catheter. However it must be pointed out that it is not necessary to perform selective pancreatic vein catheterization and sampling in all cases of endocrine pan-
creatic tumors. For example insulinoma tumors usually are solitary. Hence a positive result of clinical, laboratory, and less invasive localization procedures may be sufficient for a preoperative work-up. The multipeptide content of the tumors showed a variety ofpatterns. No conclusions could be drawn regarding the tendency for the tumors to metastasize or to follow a more malignant course. The only exceptions were the PPomas in which two patients died a few months after the diagnosis was established with, respectively, four of five additional peptides in the tumor. Possibly the presence of many peptides indicates a lesser degree of differentiation and more aggressive growth of the tumor. These two patients had extensive liver metastasis as well. Further studies are needed to substantiate this. The criteria used for the classification of endocrine tumors have changed considerably during the last 20 years. It may be based on clinical symptoms, conventional histology, ultrastructural features, immunocytochemical findings, or circulating hormone levels. Previously it was shown that pancreatic endocrine tumors may contain a variety of peptides distributed in different cell populations.23 Furthermore there can be differences in the tumor cell composition between metastasized areas and the primary tumor.24 This study also confirms previous results indicating that most of these tumors are multihormonal. Even if we have not been able to show a consistent pattern, determining the tumor peptide content (or release) may be a possible method to subclassify pancreatic endocrine tumors. Therefore it particularly important that these relatively rare tumors are managed and analyzed carefully.
References 1. Williamson RCN. Endocrine and other unusual pancreatic tumours. Current Opinion in Gastroenterology 1986; 2:746-752. 2. Prinz RA, Bermes Jr EW, Kimmel JR, Marangos PJ. Serum markers for pancreatic islet cell and intestinal carcinoid tumors: a comparison of neuro-specific enolase, (3-human chorionic gonado-
tropin and pancreatic polypeptide. Surgery 1983; 94:1019-1023. 3. Bloom SR, Polak JM, Welbourn RB. Pancreatic apudomas. World J Surg 1979; 3:587-595. 4. McGuigan JE, Wolfe MM. Secretin injection test in the diagnosis of gastrinoma. Gastroenterology 1980; 79:1324-1331. 5. Imamura M, Takahashi K, Adachi H, et al. Usefulness of selective arterial secretin injection test for localization of gastrinoma in the Z-E syndrom. Ann Surg 1987; 205:230-239. 6. Kent RB, van Heerden JA, Weiland LH. Nonfunctioning islet cell tumors. Ann Surg 1981; 193:185-190. 7. Broughan TA, Leslie JD, Soto JM, Hermann RE. Pancreatic islet cell tumors. Surgery 1986; 99:671-678. 8. Rothmund M, Ruckert K, Beyer J. Insulinome und seltene endokrine Pankreastumoren. Chirurgische Klinik und Poliklinik 1986; 57: 541-551. 9. Gorman B, Charboneau JW, James EM, et al. Benign pancreatic insulinoma: preoperative and intreaoperative sonographic localization. AJR 1986; 147:929-934. 10. Katz LB, Aufses AH, Reyfield E, Mitty H. Preoperative localization and intraoperative glucose monitoring in the management of patients with pancreatic insulinoma. Surg Gynecol Obstet 1986; 163:509-512.
MARTENSSON 614 11. Daggett PR, Goodburn EA, Kurtz AB et al. Is preoperative localization of insulinomas necessary? Lancet 1981; i:483-486. 12. Reichardt W, Ingemansson S. Selective vein catheterization for hormone assay in endocrine tumors of the pancreas. Acta Radiol Diagnosis 1980; 21:177-187. 13. Nobin A, Erhardt K, Auer G, et al. Nuclear DNA patterns and survival in metastasizing ileal carcinoids. World J Surg 1987; 11: 372-377. 14. Larsson L-I, Grimelius L, Hakansson R, et al. Mixed endocrine pancreatic tumors producing several peptide hormones. Am J Pathol 1975; 79:271-280. 15. Heitz PU, Kasper M, Kloppel G. Pancreatic endocrine tumors. Immunocytochemical analysis of 125 tumors. Hum Pathol 1982; 13:263-271. 16. Sundler F, Alumets J, Hakansson R. Majority and minority cell populations in GEP and bronchial endocrine tumours. Scand J Gastroent 1979; 14(Suppl) 53:9-13. 17. Alumets J, Falkmer S, Hakansson R, et al. Neurohormonal peptides in endocrine tumors of the pancreas, stomach, and upper small intestine: I. An immunohistochemical study of 27 cases. Ultrastruct Pathol 1983; 5:55-72. 18. Coons AH, Leduce EH, Connolly JM. Studies on antibody produc-
AND OTHERS 19.
20. 21.
22. 23.
24.
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Surg. November 1990
tion. I. A method for the histochemical demonstration ofspecific antibody and its application to a study ofthe hyperimmune rabbit. J Exp Med 1955; 102:49-60. Friesen SR. Tumors of the endocrine pancreas. N Eng J Med 1982; 306:580-590. Moertel CG, Hanley JA, Johnson LA. Streptozocin alone compared with streptozocin plus fluorouracil in the treatment of advanced islet-cell carcinoma. N Eng J Med 1980; 303:1189-1194. Wood SM, Kraenzlin ME, Adrian TE, Bloom SR. Treatment of patients with pancreatic endocrine tumours using a new longacting somatostatin analogue. Symptomatic and peptide responses. Gut 1985; 26:438-444. Eriksson B, Oberg K, Alm G, et al. Treatment of malignant endocrine pancreatic tumors with human leucocyte interferon. Cancer Treat Rep 1987; 71:31-37. Falkmer S, Martensson H, Nobin A, Sundler F. Peptide hormones in varies types of gastro-entero-pancreatic tumors: immunohistochemical patterns and evolutionary background. In Bresciani F, ed. Progress in Cancer Research Therapy 31. New York: Raven Press, 1984, pp 597-611. Galmiche JP, Chayvialle JA, Dubois PM, et al. Calcitonin-producing pancreatic somatostatinoma. Gastroenterology 1980; 78:15771583.
of
HANS MARTENSSON, M.D., PH.D.,* GERHARD BOTTCHER, M.D.,t FRANK SUNDLER, M.D., PH.D.,t and ANDERS NOBIN, M.D., PH.D.*
Endocrine pancreatic tumors contain and frequently secret neurohormonal peptides. This phenomenon can be used as a diagnostic and classifying tool. This study analyzes 31 patients operated on because of an endocrine pancreatic tumor, including the diagnostic procedures and the localization methods. In 15 insulinoma cases only 6 patients had a positive arteriography, while all 11 selective pancreatic vein samplings were positive. The immunoreactivity showed that, besides insulin, most tumors also contained other peptides. Of four gastrinoma cases the arteriography was positive in three, but the selective vein sampling localized the tumor in all. The tumors's content of peptides showed mixed patterns. In the four glucagonomas, the arteriography was positive in all and the venous sampling performed in three of the cases also was positive. In five pancreatic polypeptide-containing tumors (PP-omas) the arteriography was positive in four and sampling performed in two was positive in both. In the PP-omas the peptide pattern showed that these tumors frequently contain several peptides. We used selective pancreatic vein sampling in 21 cases with positive result in all. In the cases in which arteriography was negative, the sampling results helped the surgeon to find the tumor. The peptide pattern in the tumors varied greatly and most tumors were multihormonal. E a NDOCRINE TUMORS OF the pancreas are rare, but
when they do occur the patients may suffer considerably before diagnosis is reached and the tumor is removed. In one important aspect endocrine tumors are different from other tumors arising in the pancreas. They produce neurohormonal peptides in varying degrees, which, in many cases, can be analyzed by radioimmunoassay. X This fact makes is possible to use neurohormonal peptides as tumor markers aiding in the diagnosis by measuring elevated fasting levels in the blood.2'3 The development of stimulation and suppression tests has increased the sensibility and diagnostic reliability even
more.4'5
From the Departments of Surgery* and Histology,t Lund University, Lund, Sweden
The true incidence of endocrine pancreatic tumors is difficult to estimate because many of these tumors are silent. However the distribution of various types of islet cell tumors is documented in several series.6'7 The most common tumor is insulinoma (50% to 60%), followed by 'nonfunctioning' tumors (15% to 25%), gastrinoma (18% to 20%), and various types (7%). Once the diagnosis is established, the question arises of where the tumor is localized within the pancreas and whether it is solitary or multiple. The noninvasive preoperative localization methods of choice are sonography and computed tomographic (CT) scanning. Sonography has been shown to be positive in 63% to 90% of cases, and CT scan is positive in 45% to 90% of cases, as compiled from several studies.8"1l Arteriography is an invasive examination with an accuracy rate of 40% to 90% and a false-positive rate of about 5%.8,9,11 The abnormal release of neurohormonal peptides from the tumors has enabled their localization by sampling blood from various branches of the portal or intestinal veins. This method, however, is highly invasive and the outcome depends on the skill of the investigator. The reported results are thus poor (60% positive) when the sampling is done in the main portal branches; they are much better, however (80% to 90% positive), when a superselective sampling is made.8 Since the early 1970s we have used the superselective sampling technique for the localization of different gastrointestinal endocrine tumors and their metastasized
areas.12 Some gastroenteropancreatic endocrine tumors behave
Address reprint requests to Hans Martensson, Department of Surgery, Helsingborg Hospital, S-251 87 Helsingborg, Sweden. Accepted for publication January 10, 1990.
more aggressively than others of the same size and extent of metastasis. Therefore to obtain prognostic information, analyses of growth pattern, peptide content, and patterns
607
608
MARTENSSON AND OTHERS
of DNA distribution in carcinoid tumors have been made, but without consistent results.'3 Hence the treatment strategy of gastroenteropancreatic endocrine tumors is still surgical intervention. To achieve radicality a preoperative mapping of the tumor extent is essential. A number of investigations have shown that endocrine pancreatic tumors frequently are multihormonal, i.e., contain several neurohormonal peptides. 14-16 Such tumors are composed of multiple endocrine cell types, some of
Ann. Surg. * November 1990
which are not seen in the normal adult pancreas. For several peptides there are reports also on elevated plasma levels in patients with pancreatic endocrine tumors.'7 It is also noteworthy that the metastasized areas may contain a different set of peptides than the primary tumor. In this series we report our experience with pancreatic vein catheterization with blood sampling to localize 31 pancreatic endocrine tumors treated at our department. Because it cannot be excluded that the peptide content
FIG. 1. Sections from two insulinomas immunostained for chromogranin A (upper left [a] and upper right [bi; 350X) and for insulin (lower left [cJ and lower right [d]; 250X and IOOX). Most of the cells display chromograninand insulinfluorescence of varying intensity. In [d] in-
tenselycinsulin-immuno-
*
~~~reactive cells
occur among the more weakly stained ones.
VOl. 212.- NO. S
ENDOCRINE PANCREATIC TUMORS
609
FIG. 2. Two insulinomas immunostained for insulin (left [a] and middle [b]; 175X). The immunoreactive cells display slightly different growth patterns. Some insulinomas also contained single scattered gastrin-immunoreactive cells (right [c]; 300X).
and pattern in the tumor may be related to the aggressiveness of the disease, we re-examined all tumor specimens available with respect to their peptide content and distribution as demonstrated by immunocytochemistry. Materials and Methods The records of 31 patients operated on because of a suspected pancreatic endocrine tumor were reviewed. The analysis encompassed clinical picture, patient age, time from start of symptoms to diagnosis, diagnostic procedures, localization methods, type of operation, tumor size and number, extent of metastasis, and survival. The pancreatic vein sampling was performed using techniques previously published from our hospital.12 Tumor tissue specimens available were re-examined using immunocytochemistry with respect to the content of neurohormonal peptides and serotonin. However none of the three islet cell hyperplasias was available for reexamination. Most pancreatic specimens, or metastasized areas, were obtained at surgery and fixed on either 4% phosphate buffer (pH 7.3; 1.5 mol/L [molar]) formaldehyde or on Bouin's solution (4% formaldehyde, 1% picric acid, 0.5% acetic acid) for 18 hours. The specimens were dehydrated through a graded series of alcohol, embedded in paraffin, and sectioned at ± 5-,tm thickness. The hydrated solutions were processed for fluorescence or peroxidase indirect immunocytochemistry'8 using primary antisera to insulin, glucagon, somatostatin, pan-
creatic polypeptide (PP), gastrin, Pro-ACTH, calcitonin (CT), calcitonin gene related peptide (CGRP), PYY, Galanin, or serotonin. Results The tumors comprise 15 insulinomas, of which 3 were islet cell hyperplasias, 5 gastrinomas, 5 pancreatic polypeptide-containing tumors (PP-omas), 4 glucagonomas, and 2 unclassified tumors. The tumor classification is based on the clinical symptoms, except for PP-omas, in which the main peptide released from and/or contained in the tumor formed the basis. From one of the two unclassified tumors, specimens were not available for re-examination and the other showed no immunocytochemical
reaction. Insulinomas Of the 15 patients, there were 11 women and 4 men, with a mean age of 57 years (median, 60 years; range, 32 to 83 years). The main symptoms were disturbances in consciousness in 14 patients, fatigue in 7 patients, and weight gain in 2 patients. Most patients were diagnosed within 1 year. In two patients, however, the time from onset of symptoms to diagnosis was more than 3 years. Fasting test with monitoring of insulin-glucose ratio was performed in 12 patients, and all were positive within 24 hours (Table 1). Arteriography was undertaken in 15 patients but was positive in only six patients (Table 1). A selective pancreatic vein sampling was performed in 11 patients, which resulted in correct localization of the tu-
610
Ann. Surg. * November 1990
MARTENSSON AND OTHERS
II
11
FIG. 3. Two glucagon-containing tumors (left [a]; 175X, and middle [b]; 200X). Note the different growth pattern and the different arrangement of glucagon cells in the two tumors. At the right, [c]; 200X, a section from the tumor shown in [b] was immunostained for PP. PP-immunofluorescent cells occur scattered in the tumor.
mor in all cases (Table 1). The mean ratio ofpeptide level between a tumor-draining vein and a large vein was 18.8
to
1.
Surgery consisted of enucleation in case, tail resection in 10 cases, and Whipple's operation in case. The tumor was solitary in all the cases and the size of each tumor is listed in Table 1. All operations were considered radical, and no patient has died from an insulinoma. The results from immunocytochemical analysis are schematically shown in Table 2A. All tumors were im-
munoreactive for insulin, 2 tumors had more peptide, 2 tumors had 2 more peptides, 2 tumors had 3 more peptides, and 1 tumor had 5 more peptides (Figs. and 2). Only one tumor contained immunocytochemically demonstrable serotonin (Table 2 A).
Gastrinomas Patients with grastrinomas included two women and three men. Their mean age was 44 years (median, 42 years;
TABLE 1. Insulinomas. Some Clinical Characteristics and the Result of Localization Procedures
Patient
Pancreatic Vein Sampling Age
No.
Sex
(years)
I 2 3 4 5 6 7 8 9 10 11 12 13 14 15
F F F F F F F F F F M M M M F
67 74
82 68 52 41 40 37 32 61 60 62 72 55 62
Tumor Size (mm) 10 40 10 20 20 ? 8
Hyperplasia Hyperplasia 15 30
Hyperplasia 30 12 ?
Arteriography
CT Scan
Sonography
Fasting
+
ND
+
+
ND ND
+ + + + + + +
ND ND ND ND ND ND
+ +
+ +
+
ND ND ND
-
F, female; M, male; ND, not done; + positive localization;
-
+
ND ND ND ND ND ND ND
negative localization.
Result
Tumor Vein
Other Vein
179 ND
+
6200,uU/mL
ND
ND
+ + + + + + + +
308 7317 >400 >400 >400
+ +
ND
ND
ND
ND 170 ND
21 27 24 45 95 ND 8 ND
ND
ND
+
ND ND
+
+
+
+
ND
245 1130 704
131 145 170
Vol. 212 * No. 5
ENDOCRINE PANCREATIC TUMORS
611
TABLE 2. The Immunocytochemical Staining Pattern
A
Pro-ACTH
CT
PYY
CGRP
Galanin
Serotonin
-
-
+
-
-
-
-
-
-
_ _
-
-
-
-
_ _
+
_ _
_ _
+
-
-
-
-
+
-
-
-
ND
ND ND +
-
+
ND ND
ND
-
-
-
+
-
Insulin
Glucagon
Somatostatin
2 3 4
+ + + + + +
-
-
-
-
-
-
+
+
-
-
-
-
-
+
-
-
+
+
-
+
+
+ + +
-
+ +
+
13 14
-
-
-
-
-
-
-
-
-
-
I
-
-
-
+
-
-
-
+
-
-
2 3 4 5 3 4
+ +
+
-
+ +
-
+
_
+
-
+
-
-
-
-
-
+
+ -
ND -
ND -
_ _ ND ND
-
+ + -
_ _ ND ND
-
+ -
_ _ ND ND ND ND
+
-
5
6 7 10 11
B
C
Gastrin
Patient
-
1
+
2
-
-
4 5
+ -
_
+ -
-
+
-
+ +
D
PP
_ ND ND ND ND
ND
+
ND
ND
-
+
-
-
-
-
+
-
-
-
+
-
-
ND
ND
ND
ND
+
-
+
+
_
_
+
-
+
+ +
_
-
-
+
+
+
_
_ _
A, insulinomas; B, gastrinomas; C, glucagonomas; D, PP-omas; + positive immunoreactivity; - negative immunoreactivity; ND, not done. range, 34 to 54 years), and the main symptom was recurrent ulceration. Two patients had perforating ulcers, of which one was situated in the proximal jejunum and
the other in the proximal duodenum. Two patients had symptoms for more than 15 years, 1 patient for 3 years, and 2 patients for less than 1 year before diagnosis was established. Basal gastrin levels were increased in all patients (Table 3). A secretin provocation test was performed in three patients: all of them responding with excessive release of gastrin. Arteriography was performed in four patients, with a positive localization in three. Selective pancreatic vein sampling in all four patients localized the tumor in all (Table 3). The mean ratio of peptide level between a tumor-draining vein and a largeig vein was 4.4 to 1. In three patients a total gastrectomy was performed before the pancreatic surgery. A tail resection was performed in three patients; in one patient the tumor was enucleated, and in one patient a biopsy was performed.
One patient had a solitary tumor, three had multiple, and had a large mass in the pancreas. The diameter of the tumors ranged between 15 and 25 mm. One patient had no metastasis, two had lymph node metastasis, and two had liver metastasis. None of the patients died from the gastrinoma, and the two with liver metastasis are free from symptoms. Three of the patients were categorized as multiple endocrine neoplasia type I (MEN I). The immunocotychemical results are shown in Table 2B. One tumor contained no immunocytochemically demonstrable gastrin but five other peptides, and in another tumor only gastrin could be demonstrated. One tumor contained one more peptide and two tumors had two more peptides. None of the tumors contained serotoninimmunoreactive cells. one
Glucagonomas There were two women and two men aged 45, 63, 62, 69 years, respectively. The symptoms in all patients were
TABLE 3. Gastrinomas. Some Clinical Characteristics and the Result of Localization Procedures
Patient No.
Sex
Age (years)
1 2 3
M F M M F
52 38 42 54 34
4 5
Tumor Size (mm)
? 15 15 25 ?
Pancreatic Vein Sampling
Arteriography
CT Scan
Sonography ND ND ND ND ND
+
+
ND
ND
+ +
+
-
ND ND
F, female; M, male; ND, not done; + positive localization,
-
Secretin Test
Gastrin Result
ND
+
+
ND
ND
+ + +
negative localization.
+ +
Tumor Vein
Other Vein
ND >5000 776 2380
ND 500 424 1720
Basal Level
1100 pg/ml 587 470 8200
MARTENSSON AND OTHERS
612
Ann. Surg. * November 1990
TABLE 4. Glucanomas. Some Clinical Characteristics and the Result of Localization Procedures Patient No.
Sex
I
F M F M
2 3 4
Age (years)
63 62 45
69
Pancreatic Vein Sampling
Tumor Size (mm)
Arteriography
CT Scan
Sonography
Result
Tumor Vein
Other Vein
? 20 50 40
+ + + +
ND ND ND ND
ND ND ND -
ND + +
ND 24,000 pg/mL
ND 1540
+
550
10,000
Glucagon Basal Level
Normal 1500 87 1850
F, female; M, male; ND, not done; + positive localization; - negative localization.
diabetes and skin rash. Basal levels of circulating glucagon increased in three patients (Table 4). The time elapsing from the start of symptoms to diagnosis was 2, 4, 6, and 7 years. Arteriography was performed in all patients, and selective pancreatic vein sampling in three patients, and all were positive (Table 4). The mean ratio of peptide level between a tumor-draining vein and a large vein was 16.9 to 1. One patient had a portal occlusion that prevented portal vein catheterization. Surgery consisted of biopsy in 1, enukleation in 1, and tail resection in 2 patients. All tumors were solitary and measured 20 to 40 mm in diameter. Before operation none of the patients had signs of metastasis. At surgery one patient had a large mass in the pancreas, while the others had no signs of metastasis. However after operation liver metastasis developed in one patient and another patient has increased blood glucagon levels. Two glucagonoma tumors were available for re-examination (Fig. 3). One contained only glucagon and the other also contained somatostatin and serotonin (Table 2C). were
PP-omas Of the patients with PP-omas, there were four women and one man who were aged 55, 55, 59, 70, and 37 years, respectively. Two patients had no symptoms while three had diffuse abdominal pains. The tumors were discovered during investigation for pains or incidentally at surgery for other reasons. Basal plasma levels of PP were increased in 3 patients, normal in 1, and were not measured in 1
patient. Arteriography was performed in all patients and positive in four. Selective pancreatic vein sampling done in two of the patients and was positive in both (Table 5). The mean ratio of peptide level between a tumor-draining vein and a large vein was 4.3 to 1. Arteriography failed to produce positive results in the patients lacking increased levels of PP in blood. Two patients were operated on with the Whipple procedure, while from the others only biopsies were taken. The tumors were solitary, ranging in diameter size from 25 to 50 mm. Four patients had liver metastasis and one had lymph node metastasis. One patient died 4 years after diagnosis and three died within year. The only survivor (at 5 years) was the patient with lymph node metastasis. Four PP tumors were available for re-examination and the immunocytochemical results are shown in Table 2D. All tumors contained PP, had another 3 peptides, had 4, and had 5 more peptides. Only one tumor contained only PP. No serotonin was found in these tumors. was was
Discussion
Endocrine pancreatic tumors are rare, but when they do occur they may cause specific symptoms leading the physician to make an accurate diagnosis.'9 With modern techniques the relevant neurohormonal peptides may be identified under basal conditions and after stimulation or inhibition.'9 The combination of symptoms and peptide analysis has improved the accuracy of diagnosis. The treatment of a patient with pancreatic endocrine
TABLE 5. PP-omas. Some Clinical Characteristics and the Result of Localization Procedures
Patient No.
Sex
Age (years)
1 2 3 4 5
F F F M F
70 55 55 37 59
Pancreatic Vein Sampling
Tumor Size (mm)
Arteriography
CT Scan
Sonography
50
-
-
30 30 25 ?
+ + + +
+
+ +
ND
F, female; M, male; ND, not done, + positive localization;
+ + -
ND ND +
negative localization.
Result
Tumor Vein
Other Vein
PP Basal Level
+
51 pmol/L 299gg/mL
8 131 ND ND ND
Normal 212 ND 15OO pmol/L
+ ND ND ND
ND ND ND
30000pmol/L
Vol. 212 * No. 5
613
ENDOCRINE PANCREATIC TUMORS
tumor has been, and still is, primarily surgery with the aim of radical excision. When this is not possible, an extensive reduction of tumor mass is desirable. Generally it is agreed that medical treatment with cytotoxic agents or interferon should be used when surgery is not possible or not radical.2s22 To make surgery optimal, preoperative localization of the tumors and possible metastasized areas is needed. We used superselective pancreatic vein catheterization with blood sampling in 21 cases. In all of these cases the result was positive and helped the surgeon to find the tumor. It is noteworthy that some of the insulinomas measured 12 mm or less in size, but still the sampling provided data of peptide secretion. This is even more interesting because the corresponding arteriography in these cases was negative. The advantage with the sampling technique is that it may detect small tumors where other methods fail. Only in one case was the sampling hard to interpret despite a tumor size of 5 cm: a PP-oma patient in whom the tumor contained immunoreactive PP, insulin, and glucagon, but none of the peptides displayed elevated levels in the plasma samples. This may be explained by the fact that none of these peptides were released. One possibility to overcome this may be the use of stimulation test during the sampling,5 but so far we have not used that. Therefore an uncertain or negative result of sampling may be caused by lack of neurohormonal peptide secretion or, alternatively, by the secretion of a defect peptide or a peptide fragment not picked up by the radioimmunoassay. In the 15 insulinoma cases it is interesting to note that nine arteriographies (60%) were negative while all samplings were positive. A small tumor size is not likely to be the cause of this because some of them were large and arteriography in other small tumors was positive. One possible explanation is that the vascularity of the insulinomas may be different when compared to other endocrine pancreatic tumors, thus explaining the difference in accuracy of the arteriography. Previously we calculated the arteriovenous difference in the sampling technique for localization of gastroenteropancreatic tumors.12 The interpretation of peptide values in the pancreatic vein sampling was without problems in all patients but one in this study, whereas the peptide levels expressed as the ratio between a tumor-draining vein and a larger pancreatic or portal vein were high in all the cases. The problem case is illustrative with absolute values almost normal, but the ratio being 6.5 to 1. Therefore we stopped using the technique of measuring the arteriovenous difference. The absolute value and/or the ratio between a tumor-draining vein and a large vein seems to give the same information and the patient is relieved from one catheter. However it must be pointed out that it is not necessary to perform selective pancreatic vein catheterization and sampling in all cases of endocrine pan-
creatic tumors. For example insulinoma tumors usually are solitary. Hence a positive result of clinical, laboratory, and less invasive localization procedures may be sufficient for a preoperative work-up. The multipeptide content of the tumors showed a variety ofpatterns. No conclusions could be drawn regarding the tendency for the tumors to metastasize or to follow a more malignant course. The only exceptions were the PPomas in which two patients died a few months after the diagnosis was established with, respectively, four of five additional peptides in the tumor. Possibly the presence of many peptides indicates a lesser degree of differentiation and more aggressive growth of the tumor. These two patients had extensive liver metastasis as well. Further studies are needed to substantiate this. The criteria used for the classification of endocrine tumors have changed considerably during the last 20 years. It may be based on clinical symptoms, conventional histology, ultrastructural features, immunocytochemical findings, or circulating hormone levels. Previously it was shown that pancreatic endocrine tumors may contain a variety of peptides distributed in different cell populations.23 Furthermore there can be differences in the tumor cell composition between metastasized areas and the primary tumor.24 This study also confirms previous results indicating that most of these tumors are multihormonal. Even if we have not been able to show a consistent pattern, determining the tumor peptide content (or release) may be a possible method to subclassify pancreatic endocrine tumors. Therefore it particularly important that these relatively rare tumors are managed and analyzed carefully.
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tion. I. A method for the histochemical demonstration ofspecific antibody and its application to a study ofthe hyperimmune rabbit. J Exp Med 1955; 102:49-60. Friesen SR. Tumors of the endocrine pancreas. N Eng J Med 1982; 306:580-590. Moertel CG, Hanley JA, Johnson LA. Streptozocin alone compared with streptozocin plus fluorouracil in the treatment of advanced islet-cell carcinoma. N Eng J Med 1980; 303:1189-1194. Wood SM, Kraenzlin ME, Adrian TE, Bloom SR. Treatment of patients with pancreatic endocrine tumours using a new longacting somatostatin analogue. Symptomatic and peptide responses. Gut 1985; 26:438-444. Eriksson B, Oberg K, Alm G, et al. Treatment of malignant endocrine pancreatic tumors with human leucocyte interferon. Cancer Treat Rep 1987; 71:31-37. Falkmer S, Martensson H, Nobin A, Sundler F. Peptide hormones in varies types of gastro-entero-pancreatic tumors: immunohistochemical patterns and evolutionary background. In Bresciani F, ed. Progress in Cancer Research Therapy 31. New York: Raven Press, 1984, pp 597-611. Galmiche JP, Chayvialle JA, Dubois PM, et al. Calcitonin-producing pancreatic somatostatinoma. Gastroenterology 1980; 78:15771583.
