Ectopic hyperparathyroidism in a patient with metastatic hypernephroma

Eetopic Hyperparathyroidism in a Patient With Metastatic Hypernephroma By A. James Blair, Jr., Charles D. Hawker, and Robert D. Utiger A patient with hypercalcemia and a right renal tumor was found to have a significantly elevated serum parathyroid hormone (IPTt-i) concentration as determined by a sensitive radioimmunoassay. This IPTH could not be distinguished from either the bovine PTH standard or /PTH in serum from a patient with a parathyroid adenoma. The right renal venous serum /PTH concentration was significantly higher than the right renal arterial serum IPTH. At surgery, the patient was found to have a metastatic hypernephroma. Foilowing right nephrectomy both the

serum IPTH and serum calcium concentrations remained elevated. Terminally, the serum calcium became normal, but serum /PTH remained elevated. Radioimmunoassay of an extract of the primary tumor estimated the concentration of lPTH in the tissue at 20 nglg. Data is presented demonstrating the specificity and sensitivity of the radioimmunoassay for PTH. The data support the hypothesis that nonendocrine tumors associated with hypercalcemia without osseous metastases secrete a PTH molecule that corresponds to a precursor form of PTH.

YPERCALCEMIA associated with a nonmetastasizing hypernephroma in a patient with normal parathyroid glands was first described by Albright,’ who suggested that the tumor may have elaborated a parathyroid hormonelike substance. Plimpton and Gellhorn2 reported ten cases in which hypercalcemia was associated with malignant tumors without demonstrable destruction of bone; four of these patients had renal tumors. These authors, like Albright before them, hypothesized that these tumors had secreted a substance with an action biologically similar to that of parathyroid hormone (PTH). Munson et a1.3 demonstrated that extracts of the tumors from such patients contained PTH immunoreactive material by complement fixation techniques. They reported identifying such activity in the extracts of six different tumors, of which three were of renal origin. Berson and Yalow4 used a radioimmunoassay to measure elevated levels of PTH immunoreactive material (IPTH) in the plasma of a large number of patients with bronchogenie carcinoma, even though most of these patients were normocalcemic. Sherwood et al5 were able to measure IPTH levels in extracts of tumors from seven patients with nonendocrine tumors and hyperparathyroidism, and an

H

From the Department of Medicine, Wayne County General Hospital and the University of Michigan, Eloise, Mich., and the Deparfmenf of Medicine, University of Perms~ylvania, Phifadelphia, Pa. Received for publication July 13, 1972. Supported in part by NIH Grants AM &%%O, AM 05649, and AM 33633. A. J. Blair, Jr., M.D., Ph.D.: Associate Professor of Medicine, University of Michigan, and Direcfor, Endocrine Service, Wayne County General Hospital, Eloise, Mich. C. D. Hawker, Ph.D.: Chief Clinical Chemist, Laboratory Procedures Division of The Upjohn Company, Kalamazoo, Mich. R. D. Utiger, M.D.: Associate Professor of Medicine, University of Pe~~syZvanja, and Chief, Endocrine Section, Department of Medicine, ~ospjta~ of the Universify of Pennsylvania, Philadelphia, Pa. Mefabolism,

Vol. 22, No. 2 (February),

1973

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elevated IPTH concentration in plasma from one of these patients. Primary hyperparathyroidism was carefully excluded by postmortem examination. Recently, Roof et aL6 and Riggs et al.’ described IPTH radioimmunoassay studies with 26 and 18 patients, respectively, believed to be producing PTH ectopically from a variety of nonparathyroid tumors. In each study, two antisera with varying immunochemical specificities were used. In each study, the authors concluded that there were immunochemical differences between the hormone in serum from patients with primary hyperparathyroidism, as compared to serum from patients with nonparathyroid tumors. Riggs et al.’ further hypothesized that the PTH material produced in the latter patients corresponds to a precursor form of parathyroid hormone. They suggested that this precursor form, which would normally be converted to a smaller form of PTH, is not so converted in the patients with tlte nonparathyroid tumors. We report here a case of a patient with metastatic hypernephroma with very high levels of IPTH in the serum, an arterial-venous difference in PTH concentration across the right kidney, and a measurable concentration of IPTH in an extract of the tumor tissue. In addition, we present evidence that this radioimmunoassay cannot distinguish this ectopically produced PTH from either the IPTH in serum of patients with primary hyperparathyroidism or the standard bovine PTH. We also show the results of lPTH determinations in serum from 23 other patients with a variety of tumors producing PTH. CASE REPORT B.Z. was a 66-yr-old female referred to the Wayne County General Hospital on December 27, 1966 because of a 30 lb weight loss over the previous 2 mo. A compIete gastrointestinal workup at another hospital had revealed onIy a smaIl hiatus hernia. While in that hospital, the patient had one episode of hemoptysis, but a bronchoscopic examination together with the appropriate cytologic and bacteriotogic studies were negative. The patient had active rheumatoid arthritis for the yr preceding her admission. ill-appearing female. The vital Physical examination revealed a thin, chronically signs were normal and the blood pressure was 110/70 mm Hg. The liver edge was palpable 4 cm below the right costal margin. There was swelling and tenderness of the wrists, eIbows, and knees bilateraIly, but no appreciable limitation of motion. There was swelling and tenderness of the proximal interphalangeal joints of the right hand. There were no other significant findings on physica examination. The patient had a normochromic, normocytic anemia with a hematocrit of 22%. The serum calcium was 11.6 mg/lOO ml and serum phosphorus was 3.7 mg/lOO ml. Urinanalysis was normal. A bIood urea nitrogen (BUN) was 30 mg/lOO ml, but following hydration the BUN fell to 15 and 10 mg/lOO ml; the serum creatinine was 0.9 and 0.8 mg/lOO m1, and the creatinine clearance was normal. The serum electrolytes were normal, and a test for rheumatoid factor was positive. The alkaline phosphatase concentration was moderately elevated at 57 IU/lOO ml (normal range, 9-35 IU/lOO ml). The serum calcium was repeatedly observed to be elevated, ranging from 1X.2-13.5 mg/lOO ml. Oral cortisone, 125 mg in divided doses daily for 7 days, did not cause a fall in serum calcium. Serum phosphorus levels were genera@ normal to lower than normal, ranging from 2.3-3.2 mr?-/lo0 m1. The admission chest x-ray was normal. An intravenous pyefogram revealed changes suggestive of a tumor mass in the upper pole of the right kidney. Bilateral renal angiography showed vascular changes consistent with a tumor in the upper pole of the right kidney, and right renal phlebography showed blockage of the main renal vein by presumed tumor tissue. At the time of angiography, blood was collected from the right

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artery

and vein simultaneously

for

subsequent

ZPTH determinations.

A metastatic

bone survey was negative for metastases, as was a liver scan. A right nephrectomy was performed on February 17, 1969, and a hypernephroma was found in the upper pole of that kidney; tumor tissue was found in the renal vein and a biopsy of a para-aortic lymph node was positive for tumor. A sample of the tumor was stored at -20°C for subsequent extraction and IPTH determination. Following surgery, the patient had a slight transient fall in serum calcium (Table I), but within 1 wk postoperatively the levels were again elevated. Following an infusion of 0.8 liters of phosphate solution (0.08 M NasHI’ and 0.019 M KH&‘O,/liter), the calcium returned to normal levels and the patient was subjectively improved for a few days. Subsequently, she was given oral phosphate solution (2.4 g NaH$04 and 0.9 g NasHP04) daily, and the serum calcium remained relatively normal. The serum phosphorus tended to remain low. Two wk following surgery the chest x-ray showed diffuse fine nodules throughout the lung parenchyma, consistent with early pulmonary metastases. She slowly lost weight. Her liver progressively enlarged. Her chest x-ray showed a progression in both the size and number of the presumed metastatic nodules. She ceased to eat, gradually became moribund, and died on July 2, 1969. During the last 2 wk of her life, she did not take the oral phosphate solution, but the serum calcium levels did not become elevated. Permission for an autopsy was not obtained.

PTH ASSAY

PROCEDURE

AND

RESULTS

IPTH was measured in the serum samples collected at the time of angiography and in a series of postoperative samples (Table I) by a sensitive radioimmunoassay procedure. s PTH was also measured in an extract of the tumor tissue after extraction with 20% acetone in 1% acetic acid as previously described.g The procedure for radioimmunoassay of lPTH employed pure bovine PTH as the standard and labeled hormone and a guinea pig anti-bovine PTH serum. Antibody-bound labeled PTH and free labeled PTH were separated by a modified dextran-coated charcoal procedure. Unknown human sera were assayed by comparison to a pooled normal human serum control. Dilutions of primary hyperparathyroid sera and dilutions of parathyroid adenoma extracts gave curves indistinguishable from the standard curve obtained with bovine PTH. The method is sensitive enough to measure IPTH levels in the sera of all normal subjects tested. A mean IPTH value of 255 pg/ ml with a standard deviation of 82 pg/ml was obtained for 61 normal subjects, and the normal range for serum lPTH was considered to be the normal mean *2 SD. The results obtained for the normal subjects, for 33 surgically proven cases of primary hyperparathyroidism, for 26 cases of secondary hyperparathyroidism, and for 3 cases of hypercalcemia of nonparathyroid origin are graphically shown in Fig. 1, which is a formal discriminate analysis of serum ZPTH as a function of serum calcium. For the normal group, a highly significant negative linear correlation between calcium and IPTH was obtained (r = -0.476, p < 0.001). This correlation enables the graphic method to clearly separate primary hyperparathyroid patients from normal subjects, even though 27% of the primary hyperparathyroid patients had ZPTH values overlapping the upper end of the normal range. The method of formal discriminate analysis, which was first applied by Arnaud et al.,‘O minimizes the problem of overlap between hyperparathyroid patients and normal subjects, because the IPTH values for the hyperparathyroid patients are too high for their serum

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calcium levels. The procedure also differentiates hypercalcemia due to hyperparathyroidism from hypercalcemia due to other causes. Figure I also shows the results for 24 patients (including the patient in this report) believed to have ectopic production of PTH by nonendocrine tumors. These patients variously had tumors of the kidney, lung, esophagus, liver, or colon. Twelve had IPTH values greater than the normal range, and 8 of the remaining 12 had PTH values separated from normal by the formal discriminate analysis. Visual examination of the group of 33 primary hyperparathyroid points and the 24 points for the patients with ectopic hyperparathyroidism shows that the former generally have higher PTH values for their serum calcium levels, while the latter have generally lower ZPTH values. The primary hyperparathyroid group had a mean serum calcium of 6.33 meq/liter compared to 6.30 meq/liter for the ectopic hyperparathyroid group. However, the mean serum lPTH of the primary group was 716 pg/ml, compared to 431 pg/ml for the ectopic hyperparathyroid group. Figure 2 shows the results of radioimmunoassay of the bovine PTH standard, dilutions of serum from a surgically proven primary hyperparathyroid patient (aderioma), and dilutions of one of the serum specimens from the patient in this report (Table I, S/8/69). The fact that each gave dose responses that Table 1. Serial Postoperative Concentrations Serum Calcium, and Serum Phosphorus

Date

2/l 7/69 2/18/69 2/19/69 2/20/69 2/24/69 3/03/69 3/11/69 5/02/69 5/08/69 5/13/69 5/13/69 5/14/60 5115169 5/17/69 5/19/69 5/22/69 5/29/69 6/02/69 6/10/69 6/15/69 7/01/69

of Parathyroid Hormone (IPTH), in Peripheral Venous Blood

PTH (pg/mt)

Serum Calcium (mg/lOO ml)

Serum Phosphorus (mgA00 ml)

976 820 945 1312 3129 2416 1117 883 1219 819 863 912 673 819 2022 924 359 777 551

10.5 9.8 9.7 9.8 11.2 11.0 11.4t 12.5 12.0 12.4 11.8 7.5 8.9 9.7 7.3t 8.3 8.7 10.4

4.8 4.7 2.1 1.8 2.5 2.6 1.8t 2.5 2.8 2.9 2.8 2.8 6.4 2.9 2.8 4.0t 2.5 2.4 2.4

785

9.7

2.3

*Postoperative. tSerum calcium and phosphorus values determined of Pennsylvania. Others not marked were determined General Hospital. $2.4 g NaH2P04 and 0.9 g Na2HP04 daily by mouth.

Comment

4 1 2 3 7 14

hr p.o.* day p.o. day p.o. day p.o. day p.o. day p.o.

10 a.m. 4 p.m. Started

Stopped Stopped

oral phosphate

oral phosphate on 6/15/69

by an AutoAnalyzer by an AutoAnalyzer

therapy*

therapy

at the University at Wayne County

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Fig. 1. Serum /PTH as a function of serum calcium in normal subjects (small filled circles), pasurgically tients with proven primary hyper(large parathyroidism filled circles), patients with hypocalcemia due n 0 . to renal disease (unfilled squares), patients with hypocalcemia due to causes (filled other . and patients squares), with hypercalcemia due either to Paget’s disease . I L of bone, hypervitamino$ sis D, or metastatic dis4 2 3 5 6 7 ease (unfilled triangles). SERUM CALCIUM, MEQ 1 L The 24 patients believed to be producing PTH from nonparathyroid tumors are also shown (unfilled circles). One of these points (IPTH = 876, calcium = 7.65) was obtained for the patient in this report (B.Z.) from the right renal arterial specimen taken at the time of angiography. Dashed line represents determinations made by linear regression analysis of the 61 normal points (r = -0.476, p < 0.001). The dotted lines are parallel to the dashed line and 2 SD above and below the regression line. Solid black bars indicate the normal ranges for serum /PTH and serum calcium. (The normal points and some of hyperparathyroid points were obtained while Dr. Hawker was employed at Inter Science Institute, Los Angeles, Calif. Serum calciums for those points were determined by atomic absorption spectroscopy.)

Fig. 2. Per cent antibody-bound 1251-PTH as a function of the PTH concentration (pg/ml) for the bovine standard PTH or as a function of the concentration (PI/ml) of serum used. Squares indicate points for standard bovine PTH, unfilled circles indicate points for serum (5/8/69) from the patient in this report (B.Z.), and solid circles indicate points for serum from a surgically proven case of primary hyperparathyroidism.

PTH, PGIML 31.25 62.5

125

250

500

ICOO 2000 4000

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were super~mposible indicates that the antiserum used in this radioimmunoassay recognizes these three sources of IPTH identically. The IPTH concentration in the serum from the right renal artery was found to be 876 p&ml, and from the right renal vein, 1049 pg/ml. These determinations were made at two dilutions of serum each in duplicate in three separate experiments. The arterial-venous difference was 173 2 33 pg/ml (mean L SD), which was significant (p
The data shown in Fig. 1 demonstrate that this PTH radioimmunoassay has the ability to distinguish hyperparathyroid patients from normal subjects. The data further show that a significant portion of the 24 patients suspected of having ectopic hyperparathyroidism had elevated IPTH levels. Based on the high IPTH concentrations in the specimens in Table 1, the significant right renal arterial-venous difference in serum IPTH, and the detection of IPTH in the hypernephroma extract, we conclude that the subject of this report had nonendocrine or ectopic hyperparathyroidism. Unfortunately, an autopsy was not permitted, and it is not known whether the patient’s parathyroid glands were atrophic. In addition, the arterial-venous difference in serum IPTH could have been due to contamination of the venous specimen with tumor tissue. Nevertheless, our findings as shown in Fig. 1 support the conclusion that, in cases of ectopic hyperparathyroidism, the associated tumors do secrete PTH. This patient, unfortunately, had developed metastases before surgery, although they were not then detectable. However, it is interesting to note that the metastases were able to synthesize and secrete PTH, since the serum IPTH concentrations remained elevated until the patient’s death. In fact, the serum specimen used in the dilution comparison in Fig. 2 was obtained nearly 3 mo after surgery, and the E’TH measured in it must have been produced by the metastases. It is also of interest that, although the oral phosphate therapy had been discontinued approximately 2 wk before death, the serum calcium levels remained normal. This finding suggested that the biologic activity of the tumor-produced PTH may have been altered terminally, although IPTH was still measurable, or even that the tumor-produced PTH had impaired biologic activity. However, at the time of these observations the patient was moribund and terminal, and other factors might explain this finding. To our knowledge there have been no reports of the successful extraction and biologic assay of PTH material produced in a case of ectopic hyperparathyroidism. Such an experiment would provide the best evidence that these

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tumors do secrete biologically active PTH. Nevertheless, our experiments’ and those of others provide strong correlations between radioimmunoassay and analysis procedure of biologic activity. Indeed, the formal discriminate Arnaud et al.,i” which we have also demonstrated in this report (Fig. 1) shows a highly significant negative linear correlation between serum IPTH and serum calcium in normal subjects, strong evidence of the biologic activity of the immunologically measured hormone. Berson and Yalowg demonstrated that IPTH in human serum exhibits heterogeneity, which is immunologically detectable. Arnaud et al.ri*l” and Sherwood et all3 have used the PTH assay to identify the existence of a larger form of PTH, which is distinct from a smaller circulating form of the hormone. This larger form appears to be identical to the PTH molecule that has been isolated in several laboratories and characterized,14a15 and is used in this and several other PTH assays as standard. Riggs et al.’ have been able to differentiate these forms with the use of two antisera that have widely different specificities. One of these antisera (CH-14 M) recognizes predominantly a larger (9500 MW) form of PTH, while the other (GP-1 M) recognizes predominantly a smaller (7000 MW) form of PTH. Our antiserum has been directly compared with these antisera by Dr. Arnaud and its specificity is identical to that of antiserum CH-14 M in that it recognizes predominantly the larger form of PTH. I6 This finding is consistent with our method of production of this antiserum, which employed PTH purified by gel filtration as the immunizing antigen8 and would therefore have been expected to elicit antibodies directed against the larger form. This finding also explains the data shown in Fig. 2, which indicates that the antiserum recognizes the bovine standard PTH, the primary hyperparathyroid serum PTH, and the serum PTH from the patient in this report identically. Although our antiserum does not apparently recognize all of the immunoreactive PTH in serum, the portion that it does recognize appears to be in the serum of the patient with ectopic hyperparathyroidism as well as the serum from patients with primary hyperparathyroidism. Figure 1 also supported this conclusion, but the generally lower IPTH values for the ectopic hyperparathyroid group indicated that the primary hyperparathyroid sera contained immunoreactive components that the ectopic hyperparathyroid sera did not contain. The data, therefore, support the hypothesis of Riggs et a1.7 that nonendocrine tumors associated with hypercalcemia and hypophosphatemia produce a larger PTH molecule (precursor), and that this precursor may not be converted to a smaller form in these patients. One might reasonably expect that such a conversion must take place, because the hypercalcemia and other biochemical changes associated with ectopic hyperparathyroidism are identical to those associated with primary hyperparathyroidism. l7 However, the data could be interpreted that such a conversion took place at the receptor level, rather than in the parathyroid glands or circulation, and that serum levels did not, therefore, reflect this conversion. An alternative explanation is that in ectopic hyperparathyroidism the conversion is to a form that lacks much of the immunochemical activity but retains biologic activity. Resolution of these problems will require charac-

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terization of both the small circulating form of PTH in normal human serum and of the PTH produced in ectopic hyperparathyroidism, and the direct demonstration that ectopically produced PTH is identical to PTH produced by parathyroid glands, both chemically and biologically. REFERENCES 1. Case records of the Massachusetts General Hospital (Case 27461). New Eng. J. Med. 225~789, 1941. 2. Plimpton, C. H., and Gellhorn, A.: Hypercalcemia in malignant disease without evidence of bone destruction. Am. J. Med. 21~7.50, 1956. 3. Munson, P. L., Tashjian, A. H., Jr., and Levine, L.: Evidence for parathyroid hormone in nonparathyroid tumors associated with hypercalcemia. Cancer Res. 25:

1062,1965. 4. Berson, S. A., and Yalow,

R. S.: Parathyroid hormone in plasma in adenomatous hyperparathyroidism, uremia, and bronchogenie carcinoma. Science 154~907, 1966. 5. Sherwood, L. M., O’Riordan, J. L. H., Aurbach, G. D., and Potts, J. T., Jr., Production of parathyroid hormone by nonparathyroid tumors. J. Clin. Endocrinol. Metab. 27:140,1967. 6. Roof, B. S., Carpenter, B., Fink, D. J., and Gordan, G. S.: Some thoughts on the nature of ectopic parathyroid hormones. Am. J. Med. 50:686,1971. 7. Riggs, B. L., Arnaud, C. D., Reynolds, J. C., and Smith, L. H.: Immunologic differentiation of primary hyperparathyroidism from hyperparathvroidism due to nonparathvroid cancer. J. Clin. Invest. 50:2079,

1971. 8. Hawker,

C. D., and Utiger, R. D.: Human serum parathyroid hormone: a radioimmunoassay procedure and physiological studies. In preparation.

9. Berson, S. A., and Yalow, R. 5.: Immunochemical heterogeneity of parathyroid hormone in plasma. J. Clin. Endocrinol. Metab. 26:1037, 1968. 10. Arnaud, C. D., Tsao, H. S., and Littledike, T. : Radioimmunoassay of human parathyroid hormone in serum. J. Clin. Invest. 50:21,1971. 11. -, Tsao, H. S., and Oldham, S. B.: Native human parathyroid hormone: an immunochemical investigation. Proc. Nat. Acad. Sci. USA 67:415,1970. 12. -, Sizemore, G. W., Oldham, S. B., Fischer, J. A., Tsao, H. S., and Littledike, E. T. : Human parathyroid hormone: glandular and secreted molecular species. Am. J. Med. 50~630,1971. 13. Sherwood, L. M., Rodman, J. S., and Lundberg, W. B.: Evidence for a precursor to circulating parathyroid hormone. Proc. Nat. Acad. Sci. USA 67:1631,1970. 14. Brewer, H. B., Jr., and Ronan, R.: Bovine parathyroid hormone: amino acid sequence. Proc. Nat. Acad. Sci. USA 67: 1862.1970. 15.Niall, H. D., Keutmann,

H. T., Sauer, R., Hogan, M., Dawson, B. F., Aurbach, G. D., and Potts, J. T., Jr.: The amino acid sequence of bovine parathyroid hormone I. Hoppe Seylers

1970. 16. Arnaud,

Z. Physiol.

Chem.

35:1586,

C. D.: Unpublished data. 17. Lafferty, F. W.: Pseudohyperparathyroidism. Medicine (Baltimore) 45:247,1966.