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Radioimmunoassay of parathyroid hormone in hypercalcemic patients with malignant disease
Radioimmunoassay Hypercalcemic
of Parathyroid
Patients with Malignant Disease
RALPH C. BENSON, Jr., M.D. B. LAWRENCE RIGGS, M.D. BARBARA
M. PICKARD,
CLAUDE D. ARNAUD, Rochester.
Hormone in
Ph.D
M.D.
Minnesota
From the Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901. This investigation was supported in part by Research Grants AM12302 and CA-l 1911 from the National Institutes of Health, Public Health Service. Requests for reprints should be addressed to Dr. B. Lawrence Riggs, Mayo Clinic, Rochester, Minnesota 5590 1. Presented at the Third F. Raymond Keating, Jr., Memorial Symposium-Parathyroid Hormone, Calcitonin and Vitamin D: Clinical Considerations, Mayo Clinic, Rochester, Minnesota, September 10-12, 1973.
Serum immunoreactive parathyroid hormone (iPTH) concentration was determined in 108 unselected hypercalcemic patients with malignant disease utilizing a sensitive radioimmunoassay system that is specific for the COOH-terminal region of the parathyroid hormone (PTH) molecule. In 103 (95.3 per cent) of these patients, serum iPTH concentration was abnormal for the concomitant serum calcium level. In the 48 patients in this series with clinically evident skeletal metastasis or bone marrow involvement, serum iPTH values were similar to those in the other patients. These results do not support a recent suggestion that ectopic production, by malignant tissue, of a non-PTH osteolytic substance is at least as common as ectopic production of PTH in hypercalcemic patients with cancer. Rather, ectopic hyperparathyroidism seems to be present in the overwhelming majority of hypercalcemic patients with cancer even when clinically evident bone metastasis is present. Also, the radioimmunoassay system used in this study was of practical help in differentiating ectopic from primary hyperparathyroidism. On the basis of a lower serum iPTH value for a given serum calcium increase, ectopic hyperparathyroidism could be separated from primary hyperparathyroidism with an overlap of the two groups of 7.7 per cent. Ectopic hyperparathyroidism results from synthesis and secretion of parathyroid hormone (PTH) by nonparathyroid cancer and produces a syndrome that is indistinguishable biochemically from primary hyperparathyroidism. Because ectopic hyperparathyroidism is only slightly less common than primary hyperparathyroidism [I], and because the associated malignant tumor is often occult, the differential diagnosis of hypercalcemia frequently reduces to primary versus ectopic hyperparathyroidism. Albright in 1941 first suggested that malignant tumors could produce a PTH-like peptide [ 21. This hypothesis received strong support in 1965 [3] when immunoreactive PTH (iPTH) ‘was identified in extracts of malignant tumors from patients with hypercalcemia, hypophosphatemia and no apparent skeletal metastasis. Subsequently, others confirmed the presence of iPTH in tumor extracts [ 4-61 or in peripheral serum [4-91 from patients with this syndrome. Radioimmunoassay of serum iPTH was initially thought to be valueless in differentiating primary from ectopic hyperparathyroidism because high values were found in both syndromes [6,7]. How-
June 1974
The American
Journal
of Medicine
Volume 56
821
RADIOIMMUNOASSAY OF PTH IN MALIGNANT DISEASE-BENSON
TABLE
I
ET AL
In the present study, we assessed the incidence of ectopic hyperparathyroidism in a large, unselected series of hypercalcemic patients with malignant dis-
Diagnoses in Patients With Cancer Patients (no.)
Cancer Type Squamous cell, lung Adenocarcinoma of lung Small cell carcinoma of lung Hypernephroma Lymphoma Breast Myeloma Bladder Cancer of the esophagus Hepatoma Unknown primary
Colon Ovary Pharynx Fibrosarcoma Tongue Cancer of the penis Leukemia Osteogenic sarcoma Melanoma
-
With Diffuse
Total
Withaut Bone Metastasis
With Bone Metastasis
17
13
4
0
2
0
2
0
Marrow Involvement
METHODS
1
1
0
0
18 14 14 13 3 3
14 13 2 0 1 2
4 1 12 0 2 1
0 0 13 0 0
3 6
3 4
0 2
0 0
3 2 1 0 0 1
0 0 1 2 1 0
0 0 0 0 0 0
0 0
0 1
1 0
1
0
0
-
0
ever, in 1971, Roof and co-workers [8] and our laboratory [9] noted differences in immunologic characteristics between the PTH in the serum of patients with primary hyperparathyroidism and that in the serum of patients with ectopic hyperparathyroidism. We [9] further showed that, in 16 of 18 patients, ectopic hyperparathyroidism could be differentiated from primary hyperparathyroidism on the basis of a lower iPTH value for a given increase in serum calcium concentration. Recently, using scaled-up technics for gel filtration of plasma, we found [ 101 that the quantity of circulating COOH-terminal, biologically inactive fragments of PTH is greatly decreased in ectopic hyperparathyroidism as compared with primary hyperparathyroidism (explaining why the iPTH value is lower in ectopic hyperparathyroidism) whereas the quantity of circulating PTH eluting with or before the native PTH marker, and thus presumed to have biologic activity, was similar to that observed in primary hyperparathyroidism (explaining why both syndromes cause hypercalcemia).
a22
June 1974
The American Journal of Medicine
ease with and without skeletal metastasis to extend our previous studies on the usefulness of radioimmunoassay of PTH in differentiating primary hyperparathyroidism from ectopic hyperparathyroidism.
Volume 58
AND
MATERIALS
We studied 87 patients with surgically proved primary hyperparathyroidism and 108 unselected hypercalcemic patients with cancer with or without clinically evident bony metastasis (Table I). Serum calcium was measured by atomic absorption flame spectrophotometry (normal 8.9 to 10.1 mg/dl), serum phosphorus by an automated version of the Fiske and Subbarow [ 1I] technic (normal 2.5 to 4.5 mg/dl), and serum creatinine by an automated version of the Jaffe [ 121 reaction (normal for males 0.8 to 1.2 mg/dl, for females 0.6 to 0.9 mg/dl). Serum iPTH was measured by the radioimmunoassay described by Arnaud, Tsao and Littledike [ 131. This system uses a guinea pig antibody to porcine PTH (GP lM, 1:50,000 final dilution), 13il-labeled bovine PTH, and dextran-coated charcoal to separate antibody-bound from “free ” 1311-bovine PTH. It measures iPTH in greater than 95 per cent of serum specimens from normal adults (normal range undetectable to 40 ~1 eq/ml) and detects as little as 5 to 10 pg of highly purified human PTH (i-84). At the dilution used in these studies, GP 1M antiserum does not react with either synthetic bovine (Beckman Bioproducts) or synthetic human NH*-terminal PTH (l-34) (Ciba Geigy Co.) [14] and therefore is considered to be specific for the COOH-terminal region of the molecule [ 151. Serum iPTH values are expressed in terms of an equivalent concentration of a standard hyperparathyroid plasma. Although immunoassays have a high degree of specificity, we have previously emphasized the importance of excluding artifacts in the interpretation of results of radioimmunoassays of PTH [ 131. Because relatively low serum concentrations of iPTH are characteristic of ectopic hyperparathyroidism [9], the following studies were carried out to establish that we were in fact measuring iPTH in the serum of patients with this disease. (1) As a routine precaution, we used hypoparathyroid plasma “blanks” in our assay procedure [ 131. Assays of unknown serum samples or iPTH components from gel filtration experiments were carried out at three different concentrations. The lower limit of sensitivity of assays was established by examining the initial “bound to free” (B:F) ratios in incubation mixtures containing labeled PTH, antiPTH antibody and human hypoparathyroid serums but no unlabeled PTH. The maximal variation of any of the 102 incubation mixtures from the mean initial B:F ratio of its group was 9 per cent. Therefore, for this study, detectable iPTH was defined as the ability of unknown serum to produce a decrease in the initial B:F ratio greater than 18 per cent (twice the maximal variation). With these criteria, the limit of detectability of iPTH usually corresponded to a serum iPTH value between 6 and 9 ~1 eq/ml.
RADIOIMMUNOASSAY
TABLE II
Serum Calcium,
Phosphorus
and Creatinine -_____
OF PTH IN MALIGNANT DISEASE-BENSON
Values (Mean k SE)
_.___~
Calcium Patients
No.
Ca ricer Without bone metastasis With bone metastasis Without bone metastasis and nonazotemic With bone metastasis and nonazotemic All patients Primary hyperparathyroidism Nonazotemic * For difference t For difference
(2) Plasma roidism above
from
a patient
the limit of assay
gel filtration
with
detectability)
on a Bio-Gel
P-150
by lyophilization.
The
predominant
ponent
with
the
(l-84)
co-eluted
ectopic
iPTH value
~~~~ _____~~~
60 48 20
12.77 i 13.22 i 12.31 i
0.19* 0.23* 0.23*
3.04 ZIZ0.10+ 3.94 It 0.17* 2.74 5 0.13*
1.51 * 0.09* 2.501 z 0.32* 0.99 :iI 0.03
14
12.75 f
0.42*
3.0 + 0.11*
0.85 _t 0.05
3.45 I!Z 0.101 2.62 _t 0.06* 2.54 it 0.07*
1.96 & 0.16*1 0.97 + 0.02 0.86 i 0.01
12.97 f 0.15*t 11.58 + 0.12* 11.32 i 0.09*
hyperparathy-
time.
(just
was fractionated
by
and concentrated
immunoreactive
1311-labeled
bovine
com-
native
PTH
marker.
(3) To evaluate
the
iPTH concentration
(r@dl)
P
of 12 1.11eq/ml
column
Creatinlne
(mg/dl)
from normal, P
and with a serum
Phosphorus
(mg/dl)
108 87 60
ET AL.
that
might
be
due to basal secretion from suppressed parathyroid glands during comparable hypercalcemia, serum iPTH was assayed in five normal persons during a standard intravenous infusion of calcium. Four hours after the onset of the infusion, when the mean serum calcium was 13 mg/dl, the highest value for iPTH in any patient was 9 ~1 eq/ml. Even these levels probably represent an overestimate because some species of iPTH that are detected by GP 1M antiserum have a half-life in serum of many hours [ 161. (4) Based on these results, we considered an iPTH value to be abnormal for hypercalcemic patients with cancer only when it was greater than 10 ~1 eq/ml.
Mean
(*SE)
iPTH
was
173
calcium
correlated
with
18.41
log iPTH
~1 eq/ml.
(r =
skeletal metastasis. Mean (&SE) iPTH was 32.6 f 3.4 ~1 eq/ml. Serum calcium did not correlate with IPTH. Hypercalcemic patients with breast cancer were classified separately because they usually are responsive to corticosteroid therapy whereas patients with other tumors usually are not [ 18-201, raising the possibility of different etiologic mechanisms. Patients
.
. .
1000
.
I
*
* .
PRIMARY HPT CANCER NORMAL
RESULTS
Mean serum calcium values were higher in patients with cancer than in patients with primary hyperparathyroidism (Table II). The hypercalcemia was most severe in the group with carcinoma of the breast. Both mean serum phosphorus and mean serum creatinine levels were higher in hypercalcemic patients with cancer than in the patients with primary hyperparathyroidism. Of the 108 patients with malignant disease, 68 had azotemia; of the 87 patients with primary hyperparathyroidism, 26 had azotemia. When the patients with azotemia are excluded, mean serum phosphorus values in the two groups were significantly lower than normal. Serum iPTH values in the patients with primary hyperparathyroidism were similar to values previously reported with antiserum GP 1M [ 13,171 (Figure 1). All these patients had detectable values and all values were abnormal for the serum calcium value at that
f
0.48; P
--_-i--L
0
9
IO
II 12 13 14 15 I6 '7 SERUM CALCIUM,mg/lOOml
18
19 20
Figure 1. Relationship between serum iPTH (assayed using GP IM) and serum calcium in primary hyperparathyroid (0) and in hypercalcemic patients with cancer (0). Serum iPTH is given in terms of equivalents of a standard hyperparathyroid plasma. For a given serum calcium value, serum iPTH was lower in patients with ectopic hyperparathyroidism and was undetectable (A) in 5 of the 108 patients with cancer. Shaded region indicates area in which normal values would fall.
June 1974
The American
Journal
of Medicine
Volume
56
023
RADIOIMMUNOASSAY
>I00
OF PTH IN MALIGNANT DISEASE-BENSON
C
‘:
[
l
100 r
*[
l
.
.’ .
. .
40
t
.
t
.
I
o&f3 B IO 12 l
’
.
.
.
20
ET AL.
. .
l*
. . .
.
47
14 I6 18 IO I2 I4 SERUM CALCIUM, mg/lOOml
16
I8
Figure 2. Relationship between serum iPTH (assayed using GP IM) and serum calcium. A, hypercalcemic patients with cancer but without metastasis. B, hypercalcemic patients with cancer with metastasis. C, hypercalcemic patients with carcinoma of the breast. D, hypercalcemic patients with cancer with diffuse marrow involvement. There was no difference in mean serum iPTH among any of these groups.
with diffuse bone marrow involvement (multiple myeloma and leukemia) were also classified separately because of the possibility that hypercalcemia might be produced by destruction of bone. There was no difference in mean serum iPTH in patients with malignant disease with or without clinically evident bone metastatis, in patients with breast cancer or in patients with diffuse bone marrow involvement. Serum iPTH values plotted as a function of serum calcium in these four groups of patients are shown in Figure 2. Linear discriminant analysis, using iPTH and serum calcium values simultaneously, separated the two groups (at any serum calcium value, iPTH was lower in the ectopic group) with an overlap of only 15 of the 195 patients (7.7 per cent); 8 patients with ectopic hyperparathyroidism and 7 patients with primary hyperparathyroidism could not be separated. COMMENTS This study is the first systematic attempt to define the incidence of ectopic hyperparathyroidism by using
824
June 1974
The American Journal of Medicine
Volume 56
radioimmunoassay in a large group of unselected hypercalcemic patients with malignant disease. Theoretically, hypercalcemia in these patients might be due to any one of three mechanisms: (1) ectopic hyperparathyroidism, (2) elaboration by the tumor of an osteolytic substance other than PTH or (3) metastatic destruction of bone. Hypercalcemia would be associated with detectable and therefore abnormal serum concentrations of iPTH only with the first of these mechanisms. Consequently, a preeminent role for ectopic hyperparathyroidism in the pathogenesis of the hypercalcemia is strongly suggested by our finding that more than 95 per cent of the hypercalcemic patients with malignant disease have abnormal serum concentrations of iPTH regardless of whether or not bone metastasis is present. Powell and associates [21] recently reported that they were unable to detect iPTH in serum or tumor extracts of 11 hypercalcemic hypophosphatemic patients with cancer and no apparent bone metastasis but were able to demonstrate that these tumor extracts had osteolytic activity in a bone culture system in vitro. Based on this and previous extraction studies, they postulated that, in half of the patients thought to have ectopic hyperparathyroidism on clinical grounds, the hypercalcemia and hypophosphatemia are due to elaboration by the tumor of an osteolytic substance other than PTH. Although their report is interesting and provocative, we think that it is premature to conclude that ectopic production of a non-PTH osteolytic substance is at least as common a mechanism as ectopic hyperparathyroidism in hypercalcemic hypophosphatemic patients with malignant disease. The alternative possibility is that their failure to detect iPTH in the serum or tumors of these patients has a methodologic explanation. First, the antibody used by them may not have cross reacted well with the predominant form of iPTH produced ectopically by malignant tissue. Careful examination of the data in a previous publication [22] shows that in the critical radioimmunoassays performed by these investigators the antibody had a greater binding affinity for NH*-terminal than for COOH-terminal determinants. There is evidence [lo] that PTH precursor molecules may circulate in ectopic hyperparathyroidism, and Potts et al. [23] have reported that NH*-terminal-specific assays cross react with PTH precursors only i/5 to l/10 as well as COOH-terminal-specific assays. Second, there may have been a bias in favor of the bioassay. Because the 8 M urea used to extract the cancer tissue interfered with the immune system, only 5 to 10 ~1 of extract could be assayed. In contrast, the urea and salt in the extracts were removed by dialysis before addition to the bone cultures for bioassay, so
RADIOIMMUNOASSAY OF PTH IN MALIGNANT DISEASE--BENSON ET AL.
that
greater
quantities
of tumor
extract
could
be
used. The diagnosis of ectopic hyperparathyroidism rests heavily on the sensitivity and specificity of the radioimmunoassay system used for measuring circulating PTH. The intrinsic sensitivity of the assay system we have used is close to the ultimate that might be expected from radioimmunoassays: our assay detects almost as little unlabeled hormone added to the assays as labeled hormone. Also, our antibody cross reacts well with the components of iPTH demonstrated by gel filtration in plasma from patients with ectopic hyperparathyroidism [ lo]. In addition, we took great care to eliminate procedural artifact (see “Methods and Materials”). For these reasons, we think that there is no need to invoke mechanisms other
than
ectopic
hyperparathyroidism
to explain
the
in a small subset of patients with the syndrome. However, definitive resolution of this important issue will await extraction of PTH from tumors of patients with the syndrome by
hypercalcemia
except
perhaps
methods capable of a thousandfold or more purification. In patients with skeletal metastasis, hypercalcemia traditionally has been believed to be caused by mechanical dissolution of bone by cancer cells and the release
of calcium
and
phosphorus
into
the
circula-
This simplistic explanation fails to take into account the common clinical observation that some patients with extensive osteolytic metastasis are normocalcemic. whereas others with minimal osteolytic tion.
metastasis
are severely
hypercalcemic:.
If mechani-
cal disruption of bone by tumor is the sole mechanism, PTH secretion by the parathyroid glands should be suppressed and serum phosphorus should increase. However, two recent studies [24,25] show that hypercalcemic patients with skeletal metastasis are hypophosphatemic (when the degree of azotemia is taken into account). Our studies suggest that ectopic hyperparathyroidism plays a causal or contributory role in hypercalcemic patients with skeletal metastasis because serum iPTH values in these patients did not differ significantly from those in hypercalcemic patients with cancer but without clinically evident skeletal metastasis. We have found radioimmunoassay of PTH to be of practical help in differentiating ectopic hyperparathyroidism due to occult malignancy from primary hyperparathyroidism. We have extended our earlier observations [9] and have found that more than 90 per cent of patients in these two groups could be separated on the basis of a lower iPTH value for a given serum calcium value. The ability of our radioimmunoassay system to make this distinction is probably due to its detection of COOH-terminal, biologically inactive fragments in the circulation. The amount of these fragments is greatly decreased in ectopic hyperparathyroidism as compared with primary hyperparathyroidism [ lo]. Radioimmunoassay systems that fail to measure COOH-terminal fragments of the PTH molecule would be expected to be less helpful in differentiating the two syndromes.
REFERENCES 1. 2. 3.
4.
5.
6.
7. 8.
9.
Lafferty FW: Pseudohyperparathyroidism. Medicine (Baltimore) 45: 247, 1966. Case records of the Massachusetts General Hospital (Case 27461). N Engl J Med 225: 789, 1941. Munson PL, Tashjian AH Jr, Levine L: Evidence for parathyroid hormone in nonparathyroid tumors associated with hypercalcemia. Cancer Res 25: 1062, 1965. Sherwood LM. O’Riordan JLH, Aurbach GD, Potts JT Jr: Production of parathyroid hormone by nonparathyroid tumors. J Clin Endocrinol Metab 27: 140, 1967. Knill-Jones RP, Buckle RM, Parsons V, Calne RY, Williams R: Hypercalcemia and increased parathyroid-hormone activity in a primary hepatoma: studies before and after hepatic transplantation. N Engl J Med 282: 704, 1970. Blair AJ Jr, Hawker CD, Utiger RD: Ectopic hyperparathyroidism in a patient with metastatic hypernephroma. Metabolism 22: 147. 1973. Reiss E: Discussion. Trans Assoc Am Physicians 81: 114, 1968. Roof BS, Carpenter B, Fink DJ, Gordan GS: Some thoughts on the nature of ectopic parathyroid hormone. Am J Med 50: 686. 1971. Riggs BL, Arnaud CD, Reynolds JC, Smith LH: Immunologic differentiation of primary hyperparathyroidism from hyperparathyroidism due to nonparathyroid cancer. J Clin Invest 50: 2079, 1971.
10.
Il. 12.
13.
14.
15.
16.
17.
18.
June 1974
Benson RC. Riggs BL. Arnaud CD: lmmunoreactive forms of parathyroid hormone in primary and ectopic hyperparathyroidism (abstract). Clin Res 21: 819, 1973. Fiske CH, Subbarow Y: The calorimetric determination of phosphorus. J Biol Chem 66: 375, 1925. Jaff$ M: Ueber den Niederschlag, welchen Pikrinsaure in normalem Harn erzeugt und uber eine neue Reaction des Kreatinins. Z Physiol Chem (Hoppe-Seyler’s) 10: 391,1886. Arnaud CD. Tsao HS, Littledike T: Radioimmunoassay of human parathyroid hormone in serum. J Clin Invest 50: 21. 1971. Andreatta RH, Hartmann A, Johl A, Kamber B, Maier R, Riniker B, Rittel W, Sieber P: Synthese der Sequenz I34 von monschlichem Parat-hormon. Helv Chim Acta 56: 470, 1973. Arnaud CD, Goldsmith RS, Bordier PJ. Sizemore GW: Influence of immunoheterogeneity of circulating parathyroid hormone on results of radioimmunoassays of serum in man. Am J Med 56: 785, 1974. Silverman R. Yalow RS: Heterogeneity of parathyroid hormone: clinical and physiologic implications. J Clin Invest 52: 1958, 1973. Purnell DC, Smith LH, Scholz DA, Elveback LR, Arnaud CD: Primary hyperparathyroidism: a prospective clinical study. Am J Med 50: 670. 1971. Myers WPL: Cortisone in the treatment of hypercalcemia in
The American
Journal
of Medicine
Volume 56
625
RADIOIMMUNOASSAY
19.
20. 21.
22.
828
OF PTH IN MALIGNANT DISEASE-BENSON
ET AL.
neoplastic disease. Cancer 11: 83, 1958. Jessiman AG, Emerson K Jr, Shah RC, Moore FD: Hypercalcemia in carcinoma of the breast. Ann Surg 157: 377, 1963. Mannheinmer IH: Hypercalcemia of breast cancer. Cancer 18: 679. 1965. Powell D, Singer FR, Murray TM, Minkin C, Potts JT Jr: Nonparathyroid humoral hypercalcemia in patients with neoplastic diseases. N Engl J Med 289: 176, 1973. Segre GV, Habener JF, Powell D, Tregear GW, Potts JT Jr: Parathyroid hormone in human plasma: immunochemical characterization and biological implications. J Clin Invest
June 1974
The American
Journal of Medicine
Volume
23.
24.
25.
56
51: 3163, 1972. Potts JT Jr, Niall HD, Tregear GW, Van Rietschoten J, Habener JF, Segre GV, Leutman HT: Chemical and biologic studies of proparathyroid hormone and parathyroid hormone: analysis of hormone biosynthesis and metabolism. Mt Sinai J Med NY 40: 448, 1973. Zilva JF, Nicholson JP: Plasma phosphate and potassium levels in the hypercalcemia of malignant disease. J Clin Endocrinol Metab 36: 1019, 1973. Schussler GC, Verso MA, Nemoto T: Phosphaturia in hypercalcemic breast cancer patients. J Clin Endocrinol Metab 35: 497, 1972.
of Parathyroid
Patients with Malignant Disease
RALPH C. BENSON, Jr., M.D. B. LAWRENCE RIGGS, M.D. BARBARA
M. PICKARD,
CLAUDE D. ARNAUD, Rochester.
Hormone in
Ph.D
M.D.
Minnesota
From the Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901. This investigation was supported in part by Research Grants AM12302 and CA-l 1911 from the National Institutes of Health, Public Health Service. Requests for reprints should be addressed to Dr. B. Lawrence Riggs, Mayo Clinic, Rochester, Minnesota 5590 1. Presented at the Third F. Raymond Keating, Jr., Memorial Symposium-Parathyroid Hormone, Calcitonin and Vitamin D: Clinical Considerations, Mayo Clinic, Rochester, Minnesota, September 10-12, 1973.
Serum immunoreactive parathyroid hormone (iPTH) concentration was determined in 108 unselected hypercalcemic patients with malignant disease utilizing a sensitive radioimmunoassay system that is specific for the COOH-terminal region of the parathyroid hormone (PTH) molecule. In 103 (95.3 per cent) of these patients, serum iPTH concentration was abnormal for the concomitant serum calcium level. In the 48 patients in this series with clinically evident skeletal metastasis or bone marrow involvement, serum iPTH values were similar to those in the other patients. These results do not support a recent suggestion that ectopic production, by malignant tissue, of a non-PTH osteolytic substance is at least as common as ectopic production of PTH in hypercalcemic patients with cancer. Rather, ectopic hyperparathyroidism seems to be present in the overwhelming majority of hypercalcemic patients with cancer even when clinically evident bone metastasis is present. Also, the radioimmunoassay system used in this study was of practical help in differentiating ectopic from primary hyperparathyroidism. On the basis of a lower serum iPTH value for a given serum calcium increase, ectopic hyperparathyroidism could be separated from primary hyperparathyroidism with an overlap of the two groups of 7.7 per cent. Ectopic hyperparathyroidism results from synthesis and secretion of parathyroid hormone (PTH) by nonparathyroid cancer and produces a syndrome that is indistinguishable biochemically from primary hyperparathyroidism. Because ectopic hyperparathyroidism is only slightly less common than primary hyperparathyroidism [I], and because the associated malignant tumor is often occult, the differential diagnosis of hypercalcemia frequently reduces to primary versus ectopic hyperparathyroidism. Albright in 1941 first suggested that malignant tumors could produce a PTH-like peptide [ 21. This hypothesis received strong support in 1965 [3] when immunoreactive PTH (iPTH) ‘was identified in extracts of malignant tumors from patients with hypercalcemia, hypophosphatemia and no apparent skeletal metastasis. Subsequently, others confirmed the presence of iPTH in tumor extracts [ 4-61 or in peripheral serum [4-91 from patients with this syndrome. Radioimmunoassay of serum iPTH was initially thought to be valueless in differentiating primary from ectopic hyperparathyroidism because high values were found in both syndromes [6,7]. How-
June 1974
The American
Journal
of Medicine
Volume 56
821
RADIOIMMUNOASSAY OF PTH IN MALIGNANT DISEASE-BENSON
TABLE
I
ET AL
In the present study, we assessed the incidence of ectopic hyperparathyroidism in a large, unselected series of hypercalcemic patients with malignant dis-
Diagnoses in Patients With Cancer Patients (no.)
Cancer Type Squamous cell, lung Adenocarcinoma of lung Small cell carcinoma of lung Hypernephroma Lymphoma Breast Myeloma Bladder Cancer of the esophagus Hepatoma Unknown primary
Colon Ovary Pharynx Fibrosarcoma Tongue Cancer of the penis Leukemia Osteogenic sarcoma Melanoma
-
With Diffuse
Total
Withaut Bone Metastasis
With Bone Metastasis
17
13
4
0
2
0
2
0
Marrow Involvement
METHODS
1
1
0
0
18 14 14 13 3 3
14 13 2 0 1 2
4 1 12 0 2 1
0 0 13 0 0
3 6
3 4
0 2
0 0
3 2 1 0 0 1
0 0 1 2 1 0
0 0 0 0 0 0
0 0
0 1
1 0
1
0
0
-
0
ever, in 1971, Roof and co-workers [8] and our laboratory [9] noted differences in immunologic characteristics between the PTH in the serum of patients with primary hyperparathyroidism and that in the serum of patients with ectopic hyperparathyroidism. We [9] further showed that, in 16 of 18 patients, ectopic hyperparathyroidism could be differentiated from primary hyperparathyroidism on the basis of a lower iPTH value for a given increase in serum calcium concentration. Recently, using scaled-up technics for gel filtration of plasma, we found [ 101 that the quantity of circulating COOH-terminal, biologically inactive fragments of PTH is greatly decreased in ectopic hyperparathyroidism as compared with primary hyperparathyroidism (explaining why the iPTH value is lower in ectopic hyperparathyroidism) whereas the quantity of circulating PTH eluting with or before the native PTH marker, and thus presumed to have biologic activity, was similar to that observed in primary hyperparathyroidism (explaining why both syndromes cause hypercalcemia).
a22
June 1974
The American Journal of Medicine
ease with and without skeletal metastasis to extend our previous studies on the usefulness of radioimmunoassay of PTH in differentiating primary hyperparathyroidism from ectopic hyperparathyroidism.
Volume 58
AND
MATERIALS
We studied 87 patients with surgically proved primary hyperparathyroidism and 108 unselected hypercalcemic patients with cancer with or without clinically evident bony metastasis (Table I). Serum calcium was measured by atomic absorption flame spectrophotometry (normal 8.9 to 10.1 mg/dl), serum phosphorus by an automated version of the Fiske and Subbarow [ 1I] technic (normal 2.5 to 4.5 mg/dl), and serum creatinine by an automated version of the Jaffe [ 121 reaction (normal for males 0.8 to 1.2 mg/dl, for females 0.6 to 0.9 mg/dl). Serum iPTH was measured by the radioimmunoassay described by Arnaud, Tsao and Littledike [ 131. This system uses a guinea pig antibody to porcine PTH (GP lM, 1:50,000 final dilution), 13il-labeled bovine PTH, and dextran-coated charcoal to separate antibody-bound from “free ” 1311-bovine PTH. It measures iPTH in greater than 95 per cent of serum specimens from normal adults (normal range undetectable to 40 ~1 eq/ml) and detects as little as 5 to 10 pg of highly purified human PTH (i-84). At the dilution used in these studies, GP 1M antiserum does not react with either synthetic bovine (Beckman Bioproducts) or synthetic human NH*-terminal PTH (l-34) (Ciba Geigy Co.) [14] and therefore is considered to be specific for the COOH-terminal region of the molecule [ 151. Serum iPTH values are expressed in terms of an equivalent concentration of a standard hyperparathyroid plasma. Although immunoassays have a high degree of specificity, we have previously emphasized the importance of excluding artifacts in the interpretation of results of radioimmunoassays of PTH [ 131. Because relatively low serum concentrations of iPTH are characteristic of ectopic hyperparathyroidism [9], the following studies were carried out to establish that we were in fact measuring iPTH in the serum of patients with this disease. (1) As a routine precaution, we used hypoparathyroid plasma “blanks” in our assay procedure [ 131. Assays of unknown serum samples or iPTH components from gel filtration experiments were carried out at three different concentrations. The lower limit of sensitivity of assays was established by examining the initial “bound to free” (B:F) ratios in incubation mixtures containing labeled PTH, antiPTH antibody and human hypoparathyroid serums but no unlabeled PTH. The maximal variation of any of the 102 incubation mixtures from the mean initial B:F ratio of its group was 9 per cent. Therefore, for this study, detectable iPTH was defined as the ability of unknown serum to produce a decrease in the initial B:F ratio greater than 18 per cent (twice the maximal variation). With these criteria, the limit of detectability of iPTH usually corresponded to a serum iPTH value between 6 and 9 ~1 eq/ml.
RADIOIMMUNOASSAY
TABLE II
Serum Calcium,
Phosphorus
and Creatinine -_____
OF PTH IN MALIGNANT DISEASE-BENSON
Values (Mean k SE)
_.___~
Calcium Patients
No.
Ca ricer Without bone metastasis With bone metastasis Without bone metastasis and nonazotemic With bone metastasis and nonazotemic All patients Primary hyperparathyroidism Nonazotemic * For difference t For difference
(2) Plasma roidism above
from
a patient
the limit of assay
gel filtration
with
detectability)
on a Bio-Gel
P-150
by lyophilization.
The
predominant
ponent
with
the
(l-84)
co-eluted
ectopic
iPTH value
~~~~ _____~~~
60 48 20
12.77 i 13.22 i 12.31 i
0.19* 0.23* 0.23*
3.04 ZIZ0.10+ 3.94 It 0.17* 2.74 5 0.13*
1.51 * 0.09* 2.501 z 0.32* 0.99 :iI 0.03
14
12.75 f
0.42*
3.0 + 0.11*
0.85 _t 0.05
3.45 I!Z 0.101 2.62 _t 0.06* 2.54 it 0.07*
1.96 & 0.16*1 0.97 + 0.02 0.86 i 0.01
12.97 f 0.15*t 11.58 + 0.12* 11.32 i 0.09*
hyperparathy-
time.
(just
was fractionated
by
and concentrated
immunoreactive
1311-labeled
bovine
com-
native
PTH
marker.
(3) To evaluate
the
iPTH concentration
(r@dl)
P
of 12 1.11eq/ml
column
Creatinlne
(mg/dl)
from normal, P
and with a serum
Phosphorus
(mg/dl)
108 87 60
ET AL.
that
might
be
due to basal secretion from suppressed parathyroid glands during comparable hypercalcemia, serum iPTH was assayed in five normal persons during a standard intravenous infusion of calcium. Four hours after the onset of the infusion, when the mean serum calcium was 13 mg/dl, the highest value for iPTH in any patient was 9 ~1 eq/ml. Even these levels probably represent an overestimate because some species of iPTH that are detected by GP 1M antiserum have a half-life in serum of many hours [ 161. (4) Based on these results, we considered an iPTH value to be abnormal for hypercalcemic patients with cancer only when it was greater than 10 ~1 eq/ml.
Mean
(*SE)
iPTH
was
173
calcium
correlated
with
18.41
log iPTH
~1 eq/ml.
(r =
skeletal metastasis. Mean (&SE) iPTH was 32.6 f 3.4 ~1 eq/ml. Serum calcium did not correlate with IPTH. Hypercalcemic patients with breast cancer were classified separately because they usually are responsive to corticosteroid therapy whereas patients with other tumors usually are not [ 18-201, raising the possibility of different etiologic mechanisms. Patients
.
. .
1000
.
I
*
* .
PRIMARY HPT CANCER NORMAL
RESULTS
Mean serum calcium values were higher in patients with cancer than in patients with primary hyperparathyroidism (Table II). The hypercalcemia was most severe in the group with carcinoma of the breast. Both mean serum phosphorus and mean serum creatinine levels were higher in hypercalcemic patients with cancer than in the patients with primary hyperparathyroidism. Of the 108 patients with malignant disease, 68 had azotemia; of the 87 patients with primary hyperparathyroidism, 26 had azotemia. When the patients with azotemia are excluded, mean serum phosphorus values in the two groups were significantly lower than normal. Serum iPTH values in the patients with primary hyperparathyroidism were similar to values previously reported with antiserum GP 1M [ 13,171 (Figure 1). All these patients had detectable values and all values were abnormal for the serum calcium value at that
f
0.48; P
--_-i--L
0
9
IO
II 12 13 14 15 I6 '7 SERUM CALCIUM,mg/lOOml
18
19 20
Figure 1. Relationship between serum iPTH (assayed using GP IM) and serum calcium in primary hyperparathyroid (0) and in hypercalcemic patients with cancer (0). Serum iPTH is given in terms of equivalents of a standard hyperparathyroid plasma. For a given serum calcium value, serum iPTH was lower in patients with ectopic hyperparathyroidism and was undetectable (A) in 5 of the 108 patients with cancer. Shaded region indicates area in which normal values would fall.
June 1974
The American
Journal
of Medicine
Volume
56
023
RADIOIMMUNOASSAY
>I00
OF PTH IN MALIGNANT DISEASE-BENSON
C
‘:
[
l
100 r
*[
l
.
.’ .
. .
40
t
.
t
.
I
o&f3 B IO 12 l
’
.
.
.
20
ET AL.
. .
l*
. . .
.
47
14 I6 18 IO I2 I4 SERUM CALCIUM, mg/lOOml
16
I8
Figure 2. Relationship between serum iPTH (assayed using GP IM) and serum calcium. A, hypercalcemic patients with cancer but without metastasis. B, hypercalcemic patients with cancer with metastasis. C, hypercalcemic patients with carcinoma of the breast. D, hypercalcemic patients with cancer with diffuse marrow involvement. There was no difference in mean serum iPTH among any of these groups.
with diffuse bone marrow involvement (multiple myeloma and leukemia) were also classified separately because of the possibility that hypercalcemia might be produced by destruction of bone. There was no difference in mean serum iPTH in patients with malignant disease with or without clinically evident bone metastatis, in patients with breast cancer or in patients with diffuse bone marrow involvement. Serum iPTH values plotted as a function of serum calcium in these four groups of patients are shown in Figure 2. Linear discriminant analysis, using iPTH and serum calcium values simultaneously, separated the two groups (at any serum calcium value, iPTH was lower in the ectopic group) with an overlap of only 15 of the 195 patients (7.7 per cent); 8 patients with ectopic hyperparathyroidism and 7 patients with primary hyperparathyroidism could not be separated. COMMENTS This study is the first systematic attempt to define the incidence of ectopic hyperparathyroidism by using
824
June 1974
The American Journal of Medicine
Volume 56
radioimmunoassay in a large group of unselected hypercalcemic patients with malignant disease. Theoretically, hypercalcemia in these patients might be due to any one of three mechanisms: (1) ectopic hyperparathyroidism, (2) elaboration by the tumor of an osteolytic substance other than PTH or (3) metastatic destruction of bone. Hypercalcemia would be associated with detectable and therefore abnormal serum concentrations of iPTH only with the first of these mechanisms. Consequently, a preeminent role for ectopic hyperparathyroidism in the pathogenesis of the hypercalcemia is strongly suggested by our finding that more than 95 per cent of the hypercalcemic patients with malignant disease have abnormal serum concentrations of iPTH regardless of whether or not bone metastasis is present. Powell and associates [21] recently reported that they were unable to detect iPTH in serum or tumor extracts of 11 hypercalcemic hypophosphatemic patients with cancer and no apparent bone metastasis but were able to demonstrate that these tumor extracts had osteolytic activity in a bone culture system in vitro. Based on this and previous extraction studies, they postulated that, in half of the patients thought to have ectopic hyperparathyroidism on clinical grounds, the hypercalcemia and hypophosphatemia are due to elaboration by the tumor of an osteolytic substance other than PTH. Although their report is interesting and provocative, we think that it is premature to conclude that ectopic production of a non-PTH osteolytic substance is at least as common a mechanism as ectopic hyperparathyroidism in hypercalcemic hypophosphatemic patients with malignant disease. The alternative possibility is that their failure to detect iPTH in the serum or tumors of these patients has a methodologic explanation. First, the antibody used by them may not have cross reacted well with the predominant form of iPTH produced ectopically by malignant tissue. Careful examination of the data in a previous publication [22] shows that in the critical radioimmunoassays performed by these investigators the antibody had a greater binding affinity for NH*-terminal than for COOH-terminal determinants. There is evidence [lo] that PTH precursor molecules may circulate in ectopic hyperparathyroidism, and Potts et al. [23] have reported that NH*-terminal-specific assays cross react with PTH precursors only i/5 to l/10 as well as COOH-terminal-specific assays. Second, there may have been a bias in favor of the bioassay. Because the 8 M urea used to extract the cancer tissue interfered with the immune system, only 5 to 10 ~1 of extract could be assayed. In contrast, the urea and salt in the extracts were removed by dialysis before addition to the bone cultures for bioassay, so
RADIOIMMUNOASSAY OF PTH IN MALIGNANT DISEASE--BENSON ET AL.
that
greater
quantities
of tumor
extract
could
be
used. The diagnosis of ectopic hyperparathyroidism rests heavily on the sensitivity and specificity of the radioimmunoassay system used for measuring circulating PTH. The intrinsic sensitivity of the assay system we have used is close to the ultimate that might be expected from radioimmunoassays: our assay detects almost as little unlabeled hormone added to the assays as labeled hormone. Also, our antibody cross reacts well with the components of iPTH demonstrated by gel filtration in plasma from patients with ectopic hyperparathyroidism [ lo]. In addition, we took great care to eliminate procedural artifact (see “Methods and Materials”). For these reasons, we think that there is no need to invoke mechanisms other
than
ectopic
hyperparathyroidism
to explain
the
in a small subset of patients with the syndrome. However, definitive resolution of this important issue will await extraction of PTH from tumors of patients with the syndrome by
hypercalcemia
except
perhaps
methods capable of a thousandfold or more purification. In patients with skeletal metastasis, hypercalcemia traditionally has been believed to be caused by mechanical dissolution of bone by cancer cells and the release
of calcium
and
phosphorus
into
the
circula-
This simplistic explanation fails to take into account the common clinical observation that some patients with extensive osteolytic metastasis are normocalcemic. whereas others with minimal osteolytic tion.
metastasis
are severely
hypercalcemic:.
If mechani-
cal disruption of bone by tumor is the sole mechanism, PTH secretion by the parathyroid glands should be suppressed and serum phosphorus should increase. However, two recent studies [24,25] show that hypercalcemic patients with skeletal metastasis are hypophosphatemic (when the degree of azotemia is taken into account). Our studies suggest that ectopic hyperparathyroidism plays a causal or contributory role in hypercalcemic patients with skeletal metastasis because serum iPTH values in these patients did not differ significantly from those in hypercalcemic patients with cancer but without clinically evident skeletal metastasis. We have found radioimmunoassay of PTH to be of practical help in differentiating ectopic hyperparathyroidism due to occult malignancy from primary hyperparathyroidism. We have extended our earlier observations [9] and have found that more than 90 per cent of patients in these two groups could be separated on the basis of a lower iPTH value for a given serum calcium value. The ability of our radioimmunoassay system to make this distinction is probably due to its detection of COOH-terminal, biologically inactive fragments in the circulation. The amount of these fragments is greatly decreased in ectopic hyperparathyroidism as compared with primary hyperparathyroidism [ lo]. Radioimmunoassay systems that fail to measure COOH-terminal fragments of the PTH molecule would be expected to be less helpful in differentiating the two syndromes.
REFERENCES 1. 2. 3.
4.
5.
6.
7. 8.
9.
Lafferty FW: Pseudohyperparathyroidism. Medicine (Baltimore) 45: 247, 1966. Case records of the Massachusetts General Hospital (Case 27461). N Engl J Med 225: 789, 1941. Munson PL, Tashjian AH Jr, Levine L: Evidence for parathyroid hormone in nonparathyroid tumors associated with hypercalcemia. Cancer Res 25: 1062, 1965. Sherwood LM. O’Riordan JLH, Aurbach GD, Potts JT Jr: Production of parathyroid hormone by nonparathyroid tumors. J Clin Endocrinol Metab 27: 140, 1967. Knill-Jones RP, Buckle RM, Parsons V, Calne RY, Williams R: Hypercalcemia and increased parathyroid-hormone activity in a primary hepatoma: studies before and after hepatic transplantation. N Engl J Med 282: 704, 1970. Blair AJ Jr, Hawker CD, Utiger RD: Ectopic hyperparathyroidism in a patient with metastatic hypernephroma. Metabolism 22: 147. 1973. Reiss E: Discussion. Trans Assoc Am Physicians 81: 114, 1968. Roof BS, Carpenter B, Fink DJ, Gordan GS: Some thoughts on the nature of ectopic parathyroid hormone. Am J Med 50: 686. 1971. Riggs BL, Arnaud CD, Reynolds JC, Smith LH: Immunologic differentiation of primary hyperparathyroidism from hyperparathyroidism due to nonparathyroid cancer. J Clin Invest 50: 2079, 1971.
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June 1974
Benson RC. Riggs BL. Arnaud CD: lmmunoreactive forms of parathyroid hormone in primary and ectopic hyperparathyroidism (abstract). Clin Res 21: 819, 1973. Fiske CH, Subbarow Y: The calorimetric determination of phosphorus. J Biol Chem 66: 375, 1925. Jaff$ M: Ueber den Niederschlag, welchen Pikrinsaure in normalem Harn erzeugt und uber eine neue Reaction des Kreatinins. Z Physiol Chem (Hoppe-Seyler’s) 10: 391,1886. Arnaud CD. Tsao HS, Littledike T: Radioimmunoassay of human parathyroid hormone in serum. J Clin Invest 50: 21. 1971. Andreatta RH, Hartmann A, Johl A, Kamber B, Maier R, Riniker B, Rittel W, Sieber P: Synthese der Sequenz I34 von monschlichem Parat-hormon. Helv Chim Acta 56: 470, 1973. Arnaud CD, Goldsmith RS, Bordier PJ. Sizemore GW: Influence of immunoheterogeneity of circulating parathyroid hormone on results of radioimmunoassays of serum in man. Am J Med 56: 785, 1974. Silverman R. Yalow RS: Heterogeneity of parathyroid hormone: clinical and physiologic implications. J Clin Invest 52: 1958, 1973. Purnell DC, Smith LH, Scholz DA, Elveback LR, Arnaud CD: Primary hyperparathyroidism: a prospective clinical study. Am J Med 50: 670. 1971. Myers WPL: Cortisone in the treatment of hypercalcemia in
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OF PTH IN MALIGNANT DISEASE-BENSON
ET AL.
neoplastic disease. Cancer 11: 83, 1958. Jessiman AG, Emerson K Jr, Shah RC, Moore FD: Hypercalcemia in carcinoma of the breast. Ann Surg 157: 377, 1963. Mannheinmer IH: Hypercalcemia of breast cancer. Cancer 18: 679. 1965. Powell D, Singer FR, Murray TM, Minkin C, Potts JT Jr: Nonparathyroid humoral hypercalcemia in patients with neoplastic diseases. N Engl J Med 289: 176, 1973. Segre GV, Habener JF, Powell D, Tregear GW, Potts JT Jr: Parathyroid hormone in human plasma: immunochemical characterization and biological implications. J Clin Invest
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Journal of Medicine
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51: 3163, 1972. Potts JT Jr, Niall HD, Tregear GW, Van Rietschoten J, Habener JF, Segre GV, Leutman HT: Chemical and biologic studies of proparathyroid hormone and parathyroid hormone: analysis of hormone biosynthesis and metabolism. Mt Sinai J Med NY 40: 448, 1973. Zilva JF, Nicholson JP: Plasma phosphate and potassium levels in the hypercalcemia of malignant disease. J Clin Endocrinol Metab 36: 1019, 1973. Schussler GC, Verso MA, Nemoto T: Phosphaturia in hypercalcemic breast cancer patients. J Clin Endocrinol Metab 35: 497, 1972.