Endocrinol. Japon. 1980, S. R. No. 1, 79-86

Clinical and Hormonal Aspects of the Watery - Hypokalemia-Achlorhydria (WDHA) Syndrome due to Vasoactive Intestinal Polypeptide (VIP)-producing Tumor KEN YAMAGUCHI1, KAORU ABE1, ISAMU ADACHI1, MASAHIRO TANAKA1, MASAKAZU UEDA1, YUJI OKA1 SUMIKO MIYAKAWAl, TORU KAMEYA2 AND NOBORU YANAIHARA3 1Endocrinology and 2Pathology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104 and 3Laboratory of Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka City, Shizuoka 422

Synopsis

Three patients with the watery diarrhea-hypokalemia-achlorhydria (WDHA) syn- drome were studied. All had watery diarrhea, hypokalemia and hypercalcemia. Plasma vasoactive intestinal polypeptide (VIP) levels determined by radioimmunoassay were markedly elevated in these patients, indicating that they had VIP-producing tumors. Plasma VIP levels determined serially after the operation indicate that its determination is useful in estimating the effect of a treatment. As for multiple endocrine neoplasia type 1 (MEN1), two out of the three cases belonged to this category. Patient 1 had a brother with insulinoma, and in case 2, even though there was no family history, the autopsy revealed not only multiple tumors of the pancreas but also pituitary adenomas, chief cell hyperplasia of the

parathyroid glands, thyroid adenomas and adrenocortical adenomas. VIP and other hormones in the tumors as well as in the plasma were examined extensively in these cases. In case 1, VIP, and calcitonin were produced in the tumor and only plasma VIP levels were elevated. In case 2, with multiple tumors, tumor 1 produced VIP, glucagon, pancreatic polypeptide, gastrin and cal- citonin, and tumor 2, VIP, pancreatic polypeptide, gastrin and ƒÀ-melanocyte stimu- lating hormone. In this case, plasma VIP, pancreatic polypeptide and glucagon levels were elevated. In case 3, VIP and calcitonin were produced in the tumor, and plasma VIP and calcitonin levels were elevated. These results indicate that (1) VIP is a good tumor marker for the WDHA syndrome due to VIP-producing tumors ; (2) patients with the WDHA syndrome are sometimes associated with MEN1 ; and (3) VIP-producing tumors are multiple hor- mone-producing tumors, and VIP predominantly elevated in the plasma results in the WDHA syndrome, although other hormones such as pancreatic polypeptide,

glucagon and calcitonin are sometimes found to be elevated in plasma without contributing to the clinical features.

Vasoactive intestinal polypeptide (VIP) (Bloom et al., 1973), the so-called watery is a straight-chain polypeptide composed of diarrhea-hypokalemia-achlorhydria (WDHA) 28 amino acids with a wide variety of syndrome (Marks et al., 1967). In this paper, biological activities (Said, 1975), and its we report clinical and hormonal data overproduction by the endocrine tumors of obtained from three cases of the WDHA the pancreas or ganglioneuroblastoma is syndrome due to VIP-producing tumors of known to result in profuse watery diarrhea the pancreas. Endocrinol. Japan. 80 YAMNGUCHI et al. December 1980

ACTH and ƒÀ-MSH, the hormone detected was thought to be derived from tumor cells. Materials and Methods The tumor tissues were examined by conventional morphological techniques such as tinctorial and silver Three patients with the WDHA syndrome who staining for identifying the type of endocrine cells. were shown to have endocrine tumors of the pancreas Furthermore, the indirect immunofluorescence tech- (Kawanaka et al. , 1978 ; Yamada et al. , 1978 ; Isono nique (Kameya et al., 1977) was used to detect et al., 1979) were studied. Clinical information con- tumor cells which contained Ins, Glu, PP, VIP, Gas cerning the following items was obtained from these and CT. patients : age, sex, family history, past history, the daily volume of watery diarrhea, serum potassium and calcium levels, basal acid output of the , Results 50-g oral glucose tolerance tests (50-g O-GTT), angio- graphic studies and surgical or autopsy data. Peripheral venous blood was obtained from these Clinical and laboratory data on the three patients and treated as reported previously (Abe et patients are summarized in Table 1. They al., 1979) and plasma hormone levels were assayed were all female. The volume of watery by the radioimmunoassays for insulin (Ins), glucagon (Glu), pancreatic polypeptide (PP), VIP, gastrin diarrhea reached 5 1/day in case 2. All had (Gas), calcitonin (CT) and parathyroid hor- mone marked hypokalemia and hypercalcemia. (PTH). The basal acid output was low in cases 1 Plasma VIP levels were also studied in 13 normal and 3. Glucose tolerance was abnormal in subjects, 52 patients with uremia, 14 with liver cir- rhosis, 10 with other types of endocrine tumors of case 1. Celiac angiography revealed hyper- the pancreas including insulinoma and the Zollinger- vascular tumor stains in cases 1 and 3. Ellison syndrome, and 34 with watery diarrhea. The presence of multiple endocrine In two patients with the WDHA syndrome, plasma neoplasia type 1 (MEN1) in these patients VIP levels before and after operation were deter- mined serially. is summarized in Table 2. The family history The tumor tissues and macroscopically normal of case 1 revealed an elder brother with pancreatic tissues removed with the tumor were ob- insulinoma. No patient had a past history tained at operation (cases 1 and 2) or at autopsy suggesting the presence of MEN1. In the (cases 2 and 3). They were extracted by the method reported previously (Tanaka et al., 1979). In one laboratory data, all showed hypercalcemia. case (case 2), the tumors were multiple and the two These observations, however, did not neces- tumor tissues were examined separately. One tumor sarily mean a parathyroid lesion, as will (tumor 1) was obtained at the time of operation and the other (tumor 2) at autopsy. be mentioned later. In case 2, the autopsy The content of various hormones in the tumor revealed not only multiple tumors of the was assayed by the radioimmunoassays for C-peptide pancreas but also multiple pituitary ade- (C-P), Glu, PP, somatostatin (GIF), VIP, Gas, CT, nomas, thyroid colloid adenomas, chief cell secretin (Sec), motilin (Mot), adrenocorti- cotropic hormone (ACTH) and p-melanocyte stimu- lating hyperplasia of the parathyroid glands and hormone (p-MSH). The extracts were assayed at bilateral adrenocortical adenomas, indicat- three or more dilutions to obtain a dose response ing that this case belonged to the MEN1 curve. When a significant displacement was observed with only one dilution, the sample was considered to be undetectable. In order to decide whether or not Table 1. Clinical and laboratory data on three the hormone detected in the tumor was derived from cases of the WDHA syndrome it, the amount detected was compared with that found in the normal pancreas. In the case of a hormone that is normally present in the pancreas, such as C-P, Glu, PP, GIF and VIP, the hormone detected in the tumor was considered to be produced in the tumor cells when the values exceeded those of the normal pancreas. In the case of hormones that were not detected in the normal pancreas or, if detected, were not always present or were found only in small amounts, such as Gas, CT, Sec, Mot, S. R. No.1 VIP-PRODUCING TUMORS 81

Table 2. The presence of MEN1

Fig. 1. Plasma VIP levels plotted on a log scale in normal subjects and in patients with various dis- eases. In three patients with the WDHA syn- drome, plasma VIP levels on different occasions are plotted, and in case 1 open circles are the levels at times when watery di- arrhea was not severe.

category, although there was no family of watery diarrhea in about two-thirds of history of the disease. In case 3, the these cases. There were three cases with autopsy did not reveal other endocrine elevated plasma VIP level. One patient who lesions. To sum up, two of the three cases had a level of 360 pg/ml received a laparo- had some findings compatible with MEN1. tomy, but no tumor was found in the pan- Plasma VIP levels in various types of creas and the pancreatic tail was removed. In this patient plasma VIP levels were still patients including the three with the WDHA syndrome are shown in Fig. 1. The norml high even after the operation, and she is values are less than 100 pg/ml. In some still under careful observation. In the other patients with uremia or liver , slight two with a slightly elevated level, one was elevation of plasma VIP levels were obser- a uremic patient, and in the other the ved. In the cases with insulinoma or the etiology was unknown. The plasma VIP Zollinger-Ellison syndrome, plasma VIP levels obtained from the three patients with levels were normal. In 34 cases with watery the WDHA syndrome on different occasions diarrhea, 31 showed normal plasma VIP are plotted in Fig. 1. These values were values. They were patients with pancreatic much higher than those observed in the , lung carcinoma, medullary other diseases. An shown in the WDHA thyroid carcinoma, malignant lymphoma, case 1, the values obtained at times when parasitosis, sprue or chronic , but watery diarrhea was not severe were lower a general survey failed to reveal the etiology than the others. Endocrinol. Japon. 82 YAMAGUCHI et al. December 1980

even after the operation and the patient ex-

Case 1 pired after several days. The parts of the pancreas removed at operation and autopsy were found to contain multiple tumors. The histochemical studies showed that a large tumor (0.7•~0.7 cm, tumor 1 in Fig. 2) removed at operation was an A-cell tumor and that even larger one (2•~2 cm, tumor 2 in Fig. 2) located at the head and found at

Case 2 autopsy, was a non-A, non-B cell tumor. In addition, there were many small tumors scattered throughout the pancreas, and im- munofluorescence studies revealed that some of these were composed of insulin-producing tumor cells (insulinoma) and some others of

glucagon-producing tumor cells (gluca- gonoma). Moreover, diffuse hyperplasia of PP-producing cells was observed immuno- Case 3 histochemically at the head of the pancreas. In case 3, a large tumor occupying the body and tail of the pancreas was found at oper- ation, although it was left untouched because of invasion into neighboring tissues including the aorta. At autopsy a large tumor (12•~ 14 cm) was found and diagnosed histoche- mically as non-A, non-B cell carcinoma. Plasma VIP levels after resection of the tumor in two patients with the WDHA Fig. 2. Schematic presentations of the pancreas in syndrome were shown in Fig. 3. In case the three cases of the WDHA syndrome. 1, plasma VIP levels decreased markedly 15 min after the resection and returned to

Schematic presentations of the pancreas normal the next day. However, in case 2, in these three cases are shown in Fig. 2. plasma VIP levels remained elevated after In case 1, a distal pancreatectomy was the operation, indicating that VIP-produc- ing tumors has not been removed by the performed. After the operation, the watery diarrhea ceased, the serum calcium returned operation. to normal, and the patient resumed her The plasma levels of various hormones regular daily life. In the resected pancreas, in these three cases and their normal range two tumors were found. The larger tumor are shown in Table 3. Plasma VIP levels were markedly elevated in all three cases. (2•~3 cm) consisted of non-A, non-B tumor cells, and the microscopic tumor, which was In addition, in case 2, plasma levels of found by the pathologist, was shown by glucagon and pancreatic polypeptide were immunofluorescence to be glucagonoma. In elevated. In case 3, the plasma calcitonin case 2, a tumor (tumor 1 in Fig. 2) located level was high. The plasma gastrin level at the body was found at operation and a was elevated in cases 1 and 3, possibly distal pancreatectomy was performed. In because of the gastric hypoacidity induced this, however, the watery diarrhea continued by VIP. Plasma PTH levels were slightly S. R. No.1 VIP-PRODUCING TUMORS 83

Table 3. Plasma hormone levels in patients with the WDHA syndrome

*Pg/ml **μU/ml , .

Fig. 3. Plasma VIP levels after resection of the tumor.

Table 4. Hormone content in tumor and pancreatic tissues (ng/g wet weight)

() indicate the hormone produced by tumor.

elevated only in case 2 even in the presence produced a large amount of glucagon and of hypercalcemia. small amounts of PP and Gas. On the other The content of various hormones in the hand tumor 2, obtained at autopsy, produced tumor and normal pancreas is summarized large amounts of VIP and PP and small in Table 4, and on the basis of the criteria amounts of Gas and ƒÀ-MSH. In case 3, a described in Materials and Mothods, the large amount of VIP and a small amount hormones considered to be produced by the of CT were produced. tumor tissues are parenthesized in this table. The data from the immunofluorescence In case 1, the tumor produced a large amount study are summarized in Table 5. In case of VIP and small amounts of Gas and CT. 1 and 2, more than two hormones including In case 2, tumor 1, removed at operation, VIP were proved to be present in the tumor Endocrinol. Japon, 84 YAMAGUCHI et al. December 1980

Table 5. Detection of hormone-producing Table 6. Hormone production in patients tumor cells by immunofluorescence with the WDHA syndrome

+= Presence of hormone-producing tumor cells. -=Absence of hormone-producing tumor cells. /=Not tested.

cells, indicating that these tumors produce diarrhea, and found that three had markedly multiple hormones. In case 3, however, no elevated VIP levels associated with an hormone was stained. endocrine tumor of the pancreas. The tu- Based on the data in Tables 3, 4, and mor tissues in these cases contained large 5, hormones definitely produced in the tumor amounts of VIP. In addition, in case 1, tissues and those elevated in plasma in plasma VIP levels decreased markedly after association with their production by the removal of the 'tumor, with the disap- tumors are summarized in Table 6. All tumor pearence of watery diarrhea. On the other tissues studied were multiple hormone-pro- hand, most of the other cases had normal ducing tumors. As for plasma hormone plasma VIP levels, and neither endocrine levels, VIP was elevated in all cases, and tumors of the pancreas nor ganglion- other hormones such as pancreatic polype- euroblastoma have been found in these cases ptide, glucagon and calcitonin were some- up to now. These data indicate that the times found to be elevated as a conse- plasma VIP level is an excellent marker quence of hormone production in the tumor for diagnosis of the WDHA syndrom due tissues. to a VIP-producing tumor. From the diagnostic standpoint, it can be stressed that the presence of hypercal- Discussion cemia is important in the laboratory data on patients with the WDHA syndrome. At present, there are several reasons for Kraft et al. (1970) reported that the serum believing that VIP is a major mediator of the calcium levels were high in 16 of 21 patients WDHA syndrome. These include the obser- with this syndrome and that in half of them vations that (1) plasma VIP levels are ele- the values were greater than 12 mg/dl. The vated in patients with the WDHA syndrome fact that all three cases with the WDHA (Said and Faloona, 1975; Bloom, 1978; syndrome reported here had hypercalcemia Ebeid et al., 1978), (2) the tumor contains is compatible with those observations. a large amount of VIP (Bloom et al., 1973; The mechanisms of hypercalcemia in Said and Faloona, 1975; Yamaguchi et al., patients with the WDHA syndrome are not 1980), and (3) exogeneous porcine VIP yet clarified completely, and two possibilities produces watery diarrhea in the pig (Modlin are now considered, (1) the production of et al., 1978). hypercalcemic substances by the pan- In this study, we have examined plasma creatic tumor and (2) the coexistence of VIP levels in 37 patients with severe watery parathyroid lesions compatible with MENL. S. R. No.1 VIP-PRODUCING TUMORS 85

In these, the former is surely operating in amount than in the normal pancreas or when some cases including case 1 in this series, the hormone was found in the tumor tissue because serum calcium returned to the normal by immunofluorescence. Our criteria may level after removal of the pancreatic tumor be too strict, because it is unlikely that (Jaffe, 1978). As for the second possibility, tumor tissue contained so much normal there has been no report in the literature of pancreatic tissue. Even with these criteria, a case in which primary hyperparathyroidism however, the fact that all three cases were was proved to be the cause of hypercalcemia found to contain more than two hormones in the WDHA syndrome. In case 2 reported indicates that they were multiple hormone- here, plasma PTH was slightly elevated and producing tumors. We have reported that the autopsy revealed parathyroid hyperplasia, tumors obtained from patients with the suggesting that the hypercalcemia could Zollinger-Ellison syndrome or insulinoma be due to the concomitant parathyroid were also multiple hormone producing lesion. However, it is difficult to decide (Yamaguchi et al., 1980), indicating that whether or not the parathyroid lesions are multiple hormone production is a common the cause of hypercalcemia, because there is characteristic in endocrine tumors of the a case reported (Zollinger et al., 1968) in pancreas. which hypercalcemia recurred after removal In order to determine the relationship of hyperplastic parathyroid glands but re- between clinical features and hormones pro- turned to normal after removal of a pan- duced in the tumor, we have examined creatic tumor. Therefore, the determination plasma hormone levels and tumor hormone of plasma PTH levels is expected to be of content in the same cases. When a large value in recognizing the parathyroid lesions amount of VIP was produced by the tumor, in patients with the WDHA syndrome, it was elevated in the plasma, resulting in although the elevation of the plasma PTH watery diarrhea. In addition to VIP, some level does not necessarily mean that hyper- other hormones produced in the tumor were calcemia is due to parathyroid lesions. found to be elevated in the plasma. Glu Walsh and Roth (1976) reported pre- and PP were elevated in case 2, and CT in viously that there was no familial WDHA case 3. These additional hormones that are syndrome or one associated with other en- elevated in the plasma are considered to docrine tumors, but the brother of one be less significant in contributing to clinical patient reported here had insulinoma, and in signs and symptoms, and therefore the the other autopsy revealed the presence of clinical features in there cases were not endocrine tumors compatible with MENL. much affected. However, these biologically Recently, Hutcheon et al. (1979) also re- less active hormones may be useful as ported that the WDHA syndrome an dthe additional tumor markers in these patients. Zollinger-Ellison syndrome were indepen- It is interesting to note that there were dently found in family members. These data multiple tumors in the pancreas of patient indicate that the possible association of 2, who belonged to the MENL category, MENL should be looked for in patients with and that the hormone predominantly pro- the WDHA syndrome due to endocrine duced was different from tumor to tumor. tumors of the pancreas. From the clinical standpoint, these data indi- The presence of VIP and other hormones cate that in order to determine the definite in the tumor was examined extensively in relationship between hormone-producing these cases. The tumor production of each tumors and the clinical picture, physicians hormone was considered to be positive either should examine plasma hormone levels as when the hormone was found in a larger well as the hormone content of the tumor Endocrinol. Japon. 86 YAMAGUCHI et al. December 1980

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