Medullary carcinoma of the thyroid

Medullary carcinoma of the thyroid

PluJrmac. 11Irr. C. Vol. I. pp- 18l-20S . 1976. Specialist Subject Editors : J. M. PeriJ - ::i' o o t>J ., o -e :::: c. "E- K. E. W. 198 ...

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PluJrmac. 11Irr. C. Vol. I. pp- 18l-20S . 1976.

Specialist Subject Editors : J. M.

Peri
HERSHMAN

Printed in Great Britain

and G. A. BRAY

MEDULLARY CARCINOMA OF THE THYROID KENNETH

E. W.

MELVIN

St. Vincent Hospital and Medical Center and the Unicersity of Oregon Medical School, Portland. Oregon, U.S.A.

INTRODUCTION As a tumor of parafollicular, or C-cells, medullary carcinoma of the thyroid (MCT) differs uniquely from other tumors of the thyroid gland. In addition to the specific histologic characteristics of MCT, these differences include its familial occurrence, its frequent association with other endocrine tumors, and capacity to synthesize a variety of biologically active substances, chief amongst which is the peptide hormone, calcitonin (CT). Collectively, the tumor and its associations constitute the medullary carcinoma syndrome, which typically include MCT, pheochromocytoma, and parathyroid hyperplasia or adenoma. Included also may be a variety of other clinical, radiological, and biochemical features to be described below. Although the earlier literature (Burk, 1901; Jaquet, 1906; Stoffel, 1910; Horn, 1951; Laskowski, 1957) bears reference to thyroid tumors which were probably of this cell-type, it was not until 1959 that there appeared an authoritative account (Hazard et al., 1959) of the pathological and clinical characteristics of this solid thyroid tumor with amyloid stroma, for which the name medullary carcinoma was proposed. Subsequently the extensive investigation and progressive recognition of the many features of the medullary carcinoma syndrome has provided the basis for a number of recent reviews of this subject (Steiner et al., 1958; Melvin et al., 1972; Ljungberg, 1972; Hill et al., 1973; Keiser et al., 1973). In the discussion which follows, the MCT syndrome will be described briefly and particular emphasis given the endocrine and metabolic aspects of the tumor. THE TUMOR With the improved methods of diagnosis now available, the true incidence of MCT may prove to be higher than suspected previously. Present data suggest that MCT comprises 5-12 per cent of all thyroid carcinomas (Bertoli et al., 1%7; Steiner et al., 1%8; Robbins, 1%9; Ljungberg, 1972; Hill et al., 1973). Both sporadic and familial forms occur, the latter being transmitted as an autosomal dominant trait (Schimke and Hartmann, 1%5; Ljungberg et al., 1%7; Ljungberg, 1972; Block et al., 1%7; Steiner et al., 1%8; Melvin et al., 1971; Jackson et al., 1973; Hill et al., 1973; Keiser et al., 1973). Grossly, the tumor is usually situated in the upper or middle thirds of the lateral lobe of the thyroid and is bilateral in virtually all familial cases (Melvin et al., 1971; Ljungberg, 1972; Hill et al., 1973; Keiser et al., 1973), (Fig. 1). Bilateral involvement of the thyroid is common also in sporadic cases, but occurs less frequently than in the familial form of the disease. The rare instance of unilateral familial tumor has been reported (Gordon et al., 1973). In familial cases, more than sporadic, the tumor is frequently multicentric, scattered foci occurring throughout the thyroid gland (Melvin et al., 1972). Microscopically, the tumor had a solid configuration of polyhedral or spindle-shaped cells, interspersed with a variable amount of fibrous stroma. Deposits of amyloid within the tumor, and localized to the tumor, are the histological hallmark of MCT and occur in virtually all cases. It seems probable that the amyloid is formed initially within the tumor cells (Albores-Saavedra et al., 1%4; Ljungberg, 1972). Evidence based upon 183

184

K. E. W.

MELVIN

FIG. t. Sagittal section through the thyroid gland (above) and thymus (below) from a case of familial MeT, showing bilateral medullary thyroid carcinoma (M) and gross enlargement of a parathyroid gland (P) which lay in the thymus and which histologically showed chief cell hyperplasia. From Melvin et al. (1972), by permission.

immunofluorescent studies suggests that the amyloid in MCT may be a polymerized form of the peptide hormone, calcitonin (Wolfe, H. J., personal communication; Tateishi et al., 1972; Pearse et al., 1972; Meyer et al., 1973). Calcification within the tumor is common. Histochemically, the tumor cells may display chromaffinity and argentaffinity, indicating the presence of monoamines (Falck et al., 1%8). By means of immunocytochemical techniques, the cells of MCT have been shown to consistently contain calcitonin (CT) (Bussolati et al., 1%9; Wolfe et al., 1973). Appropriate to its cell of origin, MCT is a CT-secreting tumor (Melvin and Tashjian, 1%8; Meyer and Abdel-Bari, 1%8; Tashjian and Melvin, 1%8; Cunliffe et al., 1%8; Milhand et al., 1%8). This is a consistent feature of every case thus studied and is of great practical significance in the diagnosis of this disease (Melvin et al., 1971; Jackson et al., 1973; Keiser et al., 1973; Wolfe et al., 1973). Most recently, a condition of C-cell hyperplasia has been identified in younger members of a kindred affected by a high incidence of familial MCT (Wolfe et al., 1973). Presumably, this represents a pre-cancerous stage of the disease, manifest preoperatively by an elevated serum CT level, and demonstrable histologically by means of immunocytochemical techniques. Clinically, MCT tends to follow a relatively indolent course, but this may be quite variable. The youngest case on record (Mandelstam et al., 1970) is that of a 4!-year-old boy, already with cervical metastases. Patients have been known to survive 20 years with known metastatic disease, while in others the course has been of rapid dissemination and death within months of diagnosis. THE SYNDROME MCT, more often of the familial form than of the sporadic, is frequently accompanied by pheochromocytoma-usually bilateral, and the hyperplasia or adenoma of the parathyroid glands. Other, less frequent, associations include multiple mucosal neuromata, Marfanoid habitus, pes cavus, diffuse ganglioneuromatosis of the intestinal tract, diverticulosis, peptic ulceration, and gynecomastia (Fig. 2). Diarrhea is a common

Medullary carcinoma of the thyroid

185

FIG. 2. Patient with medullary thyroid carcinoma (sporadic) bilateral pheochromocytomas, Marfanoid habitus, multiple mucosal neuromata, and gynecomastia. From Melvin et al. (1972), by permission.

symptom, usually reflecting advanced disease, and is humorally mediated. Doctrine aspects of the syndrome are characterized by the synthesis and secretion by the tumor of CT, and of the enzymes are different histaminase (Daylin et al., 1970, 1972), neither of which appear to exert any significant metabolic effect. Occasionally there is ectopic production of ACTH by the tumor, resulting in Cushing's syndrome (Melvin et al., 1970; Donahower et al., 1968). The tumors contain a high concentration of dopa decarboxylase (Atkins et al., 1973), and in some cases, serotonon (Moertel et al., 1965), and prostaglandins (Williams et al., 1968). PHEOCHROMOCYTOMA

Prior to the recognition of MeT as a distinct clinical entity, an increased incidence of thyroid carcinoma had been noted in association with pheochromocytoma (DeCourcy and DeCourcy, 1952; Sipple, 1961). It is now established that this association is specifically with MCT (Williams, 1965;for review, Steiner et al., 1968).In familial cases of MCT approximately 50 per cent can be expected to have associated pheochromocytomas (for review, Ljungberg, 1972). The incidence of pheochromocytoma in sporadic cases of MCT appears to be much lower, and of the order of 10 per cent. Irrespective of whether the thyroid tumor is familial or sporadic, associated pheochromocytomas are bilateral in over 60 per cent of cases (Finegold and Haddad, 1963; Manning et al., 1963; Nourok, 1964; Schimke and Hartmann, 1965; Williams, 1965; Steiner et al., 1%8; Melvin et al., 1972; Ljungberg, 1972). In non-familial cases of pheochromocytoma unrelated to MeT, the incidence of bilateral tumors is only 5 per cent. The frequency with which pheochromocytomas occur bilaterally in the MCT

186

K. E. W. MELVIN

syndrome is of obvious importance in determining the surgical approach to this condition. In general, pheochromocytomas occurring in association with MCT tend to be relatively inactive and should be sought by means of measurement of the urinary vanilmandelic acid (VMA), total catecholamines, and metanephrines. In some cases, however, highly active pheochromocytomas are the presenting feature of the syndrome with the thyroid carcinoma diagnosed incidentally. In cases with positive biochemical evidence of pheochromocytoma, preoperative localization by means of adrenal angiography may be useful. No case of extra-adrenal pheochromocytoma has been recorded. Occasionally, pheochromocytoma may occur without an associated thyroid tumor, as the only manifestation of the familial syndrome (Melvin et al., 1972). Just as the thyroid tumors are frequently multifocal, so also are the pheochromocytomas (Fig. 3). In two instances, the pheochromocytoma was found to contain CT (Voelkel et aI., 1973). This latter is in keeping with the finding of calcitonin-like material in the adrenal medulla of pigs (Kaplan et al., 1970). It is of interest that the CT molecule present in the pheochromocytomas and mucosal neuroma, although biologically and immunologically similar to CT, was shown to be larger by gel filtration. It must be emphasized that the production of CT by the adrenal and neural tumors is a rare event, but that it occurs at all is important evidence of shared functional similarities between the thyroid C-cell, adrenal medulla, and peripheral neural tissue. Similar evidence for a shared functional relationship between thyroid C-cells, MCT, and pheochromocytoma is provided by the recent demonstration of dopa-decarboxylase in MCT (Atkins et al., 1973), similar to that found in the adrenal medulla, and thyroid C-cells (Hankanson et al., 1971). An interesting physical finding in some patients with pheochromocytoma and MCT, but not in cases of MCT alone, is that of medullated corneal nerves which may be visualized readily with a slit-lamp (Gorlin et al., 1968). PARATHYROID ABNORMALITIES

At least sixty-five instances of parathyroid hyperplasia or adenoma have been reported in assocation with familial or sporadic MCT (for review, Steiner et al., 1968; Melvin et al., 1972; Ljungberg, 1972; Keiser et al., 1973; Hill et al., 1973),(Fig. 1). Since the association between MCT and abnormalities of the parathyroid glands was

FIG. 3. Section through left adrenal gland from a patient with familial MCT and bilateral, multifocal pheochromocytomas. Shown are two pheochromocytomas (P) within the left adren al medulla. Attenuated rim of adrenal cortex (C) is seen on lower left. Centimeter scale. From Melvin et al. (1972),by permission.

Medullary carcinoma of the thyroid

187

recognized only recently, the true incidence of this association remains to be determined. In two larger series in which this association was sought, the incidence of histological evidence of parathyroid hyperfunction in cases of familial MCT was 83 per cent (Melvin et al., 1972) and 64 per cent (Keiser et al., 1973). In these series, twenty-eight patients had parathyroid hyperplasia, and one an adenoma. Of these, hypercalcemia was present in only nine. It is evident, therefore, that parathyroid hyperfunction occurs very frequently in association with MCT, particularly with the familial form of the disease. Although definite parathyroid adenomata has been reported, the predominant histological change is hyperplasia. The majority of such patients are not hypercalcemic. The question arises as to whether the hyperparathyroidism occurring in association with MCT is a genetically determined event, or whether it arises as a complication of the hypercalcitoninemia which consistently accompanies MCT. The former of the two possibilities appears to be the more likely, since elevated serum levels of parathyroid hormone (PTH) have been found in younger members of a kindred affected by MCT, in the absence of elevated serum levels of calcitonin (Fig. 4). The clinical and hormonal features of twelve cases of familial MCT are summarized in Table 1. HORMONE AND ENZYME SYNTHESIS CALCITONIN

CT is a peptide hormone having a hypocalcemic and hypophosphatemic effect as a result of direct inhibition of bone resorption. In most species, the hormone is secreted by cells situated within the ultimobranchial bodies, or in the thyroid gland. Based upon their function of storing and secreting CT, these cells have been termed C-cells (Pearse, 1966). Under normal circumstance, C-cells occur extremely sparsely within the human thyroid and in view of their anatomic relationship to the thyroid follicles have been referred to alternatively as parafollicular cells (Teitelbaum et al., 1971). In lower species, CT is concerned with calcium homeostasis, including protection against acute hypercalcemia. Secretion of the hormone is stimulated by an increase in the serum concentration of calcium. NORMALS

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FIG. 4. Serum parathyroid hormone (PTH) levels, as measured by radioimmunoassay, in 177 fasting, normal control subjects, twenty nine patients with familial medullary thyroid carcinoma (MCT) and ninety eight of their relatives with normal serum calcitonin (CT) values, and fifteen cases of sporadic MCT. As denoted by the broken line, the assay detects 0.20 ng of bovine PTH equivalents per milliliter of human serum. Normal values range from undetectable «0.20 ng/ml), up to 0.60 ng/rnl. In none of the subjects with proven familial MCT were serum PTH values undetectable, these ranging up to 2.7 ng/ml. Of considerable interest is the finding of serum PTH levels in excess of 0.6 ngfrnl in approximately 40 per cent of the J-kindred with presently normal serum CT values (i.e. no evidence of MCT) and among similarly normal relatives of other familial cases of MCT. Serum PTH levels were consistently normal in all the patients with sporadic (nonfamilial) MCT. From Melvin et al. (1972), by permission.

64 37

34 24 30 36 27 39 25 26

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M M M

Sex

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(Not op erated on) (Not op erated on)

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Medull ar y carcinom a Right Left

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0.45 0.99 0.55 0.79 0.40 0.30 1.6 <0.30

0.40 1.8

0.60 0.97 0.65

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7.0 29.0

3.8 6.3 5.9

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930 255 500

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+ + + + +

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Postoperati ve calcium infu sion test s§

"Normal basal level s in 140 control subjects, <0.38 ng/ml; 4-hr column designates value s at the end of 4-hr calcium-infusion test (level s in control subj ects, <0.55 ng/ml). tCalciton in content of norm al adult human thyroid gland. 0.05-0.25 MRC U/g, fre sh weight ; result s given determin ed by biologic assay (mean sta ndard-erro r limits of es timates, 15 per cent). ;Range of three to eight separat e sa mples (ra nge in normal control subjects , from und etectable levels to 0.60 ng [bovine equivalents]/ml). §- indicates low or undetectable se ru m calcitonin levels after operation that did not rise significantly during 4-hr calcium-infusion test; + indicate s high basal seru m calcitonin an d abnormal rise with calcium infus ion. ~Tu m o r < I mm in diameter; ent ire spe cimen taken for histologic exam ination.

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Age (years )

Serum parathyroid hormone (ng/m!);

TABLE I. Summary oj Cllnical and Hormone -assay Data in Thirteen members 0/ a K indred witll Familial MCTor Pheochromocytoma or Both. From Melvin et at. (1971), by Permission

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Medullary carcinoma of the thyroid

189

The suggestion that MCT might represent a tumor of Cvcells (Williams, 1966) has been substantiated. Ultrastructural similarities have been demonstrated between MCT and C-cells (Meyer and Abdel-Bari, 1968;Hill et al., 1973). MCT has been established as a calcitonin-secreting tumor (Melvin and Tashjian, 1968; Tashjian and Melvin, 1968' Meyer and Abdel-Bari, 1968; Cunliffe et al., 1968; Milhand et al., 1%8; Dube et al.: 1969; Woodhouse et al., 1969; Johnston et al., 1970; Deftos et al., 1971; Melvin et al., 1972; Jackson et al., 1973; Keiser et al., 1973),(Fig. 5). The secretion of CT by MCT is stimulated by induced hypercalcemia (Melvin et al., 1970a; Melvin et al., 1972), a response shared also by Cvcells. MCT and C-ceUs share similar immunocytochemical staining characteristics (Kalina et al., 1970; Wolfe et al., 1973), based upon their CT-content. A precancerous condition of C-cell hyperplasia has been defined in subjects genetically susceptible to familial MCT (Wolfe et al., 1973).

Diagnostic Value of CT The development of a sensitive radioimmunoassay for human CT (Tashjian et al., 1970, 1972) has permitted measurement of this hormone in the serum and urine of human subjects. As measured by this assay, the majority of normal subjects have basal serum CT levels of < 0.10 ng/ml, with some individuals having detectable levels up to 0.38 ng/ml. In response to an intravenous calcium infusion, some but not all normal subjects increase their serum CT levels to two- to three-fold, never exceeding 0.55 ng/ml (Melvin et al., 1972). By contrast, the majority of patients with MCT have resting levels of serum CT> 0.38 ng/ml, and in all such patients the serum CT levels exceed 1.0 ng/ml in response to a calcium infusion, showing as much as a fifty-sevenfold increase over resting levels (Fig. 6). Measurement of the urinary CT shows changes corresponding to those occurring in the serum and may provide useful confirmatory data in patients with marginally abnormal serum values. Recent studies have shown the usefulness of CT-assay in the epidemiological study of large kindreds affected by MCT, and the early diagnosis of the occult tumor (Melvin >1000 1000

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FIG. 5. Basal serum calcitonin (Cl) values in sixty patients with proven medullary thyroid carcinoma (MCT). In the left column are plotted values from pat ients with clinically obviou s tumor; values in right column were obtained from patients in whom, as a result of surgery , tumor tissue was not clinically evident. High serum CT values in ten of the latter probably reflect undetected, residual tumor. Broken line represents the limit of sensitivity of the assay. Not log scale on the ordinate. From Melvin et al. (1972),by permission.

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190

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FIG. 6. Response of serum calcitonin (C'T) to intravenous infusions of calcium and glucagon in patients with medullary thyroid carcinoma. Basal serum CT values were plotted as 100 per cent; values at the end of the 4-hr infusion were plotted to the right of the connecting line in each panel, as percentage of basal value. Values below the broken line represent a decrease in serum CT in response to the infusion; values above the line, an increase. From Melvin et al. (1972), by permission.

et al., 1971; Jackson et al., 1973; Keiser et al., 1973). Of the total of sixty-six cases of MCT diagnosed in the three affected kindreds comprising these studies, abnormal CT levels were the only indication of disease in twenty-six. The genealogy of one such kindred is shown in Fig. 7, in which MCT has been diagnosed in nineteen, C-cell hyperplasia in two, and pheochromocytoma in six. In thirteen of these patients, the thyroid gland was normal to palpation and radioisotopic scanning, and elevated serum and urine CT values were the only indication for surgical exploration. Bilateral MCT was found in eleven, and C-ceIl hyperplasia (Wolfe et al., 1973) in two. Studies such as these emphasize the importance of screening at regular intervals all relatives of affected individuals for familial disease.

NORMAL o MALE FEMALE

o

MEDULLARY CARCINOMA

PHEOCHROMOCYTOMA

o

()

1I

IJ

PENDiNG

D

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FI~. 7. Genealogy of a kindred with familial MCT. Decreased subjects are indicated by an oblIque stroke. The fourth generation, comprising fifty four children, has been omitted for the sake of clarity, since none presently have clinical or laboratory evidence of MCT or pheochromocytoma. From Melvin et al. (1971), by permission. Normal: D, male; 0, female. Medullary carcinoma: D, male; (), female. Pheochromocytoma: D, male; (), female. Medullary carcinoma plus pheoc?romocytoma: D, male; (), female. P, is the primary propositus, and P, is the secondary propositus. The presence or absence of MCT in the original female member of this family remains uncertain.

Medullary carcinoma of the thyroid

191

Thus the calcium infusion test with CT assays provides a mean of diagnosis of MCT with a specificity and reliability unmatched by any other serologic test presently used in the diagnosis of cancer (Year Book of Cancer, 1973). More recently, the validity and sensitivity of this test has been demonstrated in the identification of precancerous C-cell hyperplasia in three young children, nineteen of whose relatives suffered from MCT (Wolfe et al., 1973). In each of these three individuals, serial calcium infusion tests had shown a progressive rise in serum CT values from normal to abnormal levels over a period of 2-3 years (Fig. 8). Although in each instance the stimulated serum CT values were less than those observed in previously established cases of MCT, the development of even' mildly abnormal values in the context of such a strong family history justified thyroidectomy. Serial sections of the thyroid glands in each case revealed no tumors, but by means of immunocytochemical techniques and geographic assays of CT content of extracted tissue it was demonstrated that the middle and upper portions of the lateral thyroid lobes showed marked increases and clustering of calcitonin-containing cells in comparison with normal thyroid glands (Figs. 9, 10). It is of interest that the maximum concentration of C-cells in these cases occurred in the same anatomic site as the bilateral tumors in other familial cases of MCT. In patients with clinically overt MCT, serial measurement of the serum CT levels offers an objective parameter by which to evaluate the response to a given modality of treatment. High serum levels of CT preoperatively fall to normal postoperatively, upon total excision of the tumor. Persistence of elevated serum levels postoperatively is indicative of residual or metastatic tumor. Other Secretagogues of CT

There is considerable evidence that the secretion of CT by the mammalian C-cell involves activation of adenyl cyclase and generation of cyclic AMP (for review, Care et al., 1971). Many of the metabolic effects of glucagon are mediated by cyclic AMP (Robison et al., 1%8). In experimental animals, the secretion of CT is stimulated by the infusion of glucagon (Care et al., 1970); the gastrointestinal hormones, gastrin and pancreozymin (Care et al., 1972), catecholamines (Care et al., 1971), and exogenous cyclic AMP (Care et al., 1970). In patients with MCT, the secretion of CT is stimulated by the intravenous infusion of glucagon (Fig. 6), and the synthetic analogue of gastrin, pentagastrin (Gray and Munson, 1%9). As secretagogues, however, these substances do not appear on present evidence to be as potent as induced hypercalcemia. In one patient studied (Melvin et al., 1972) a- and f3-adrenergic blockade each resulted in augmentation of the secretory J.T.

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FIG. 8. Concentrations of calcitonin in serum before (left) and at the end (right) of serial calcium infusion tests in three young members of kindreds affected by familial MCT. The progressive evolution of abnormal CT values over two years Observation led to thyroidectomy, with the demonstration of C-cell hyperplasia. The lower dashed line gives the sensitivity of the assay. The upper dashed line gives the maximum upper limit of basal serum CT in normal subjects. No value greater than 0.55 ng per mI has been measured in over 300 normal subjects during a calcium-infusion test. The open circles give postoperative data. From Wolfe et al. (1973),by permission.

K. E. W.

192

MELVIN

FIG.9. Section of the thyroid gland from one of the subjects whose serum CT data are shown in Fig. 8. Clusters of polygonal to spindle-shaped cells are shown in a predominantly parafollicular location (hematoxylin and eosin stain x 400 before reproduction). These cells were confirmed by immunocytochemical techniques to be C-cells. From Wolfe et al. (1973),by permission. THYRO 0

CASE 2

-26 22-

-

-

-17

20

FIG. 10. Calcitonin content of sections of a normal human thyroid gland and of the gland from a patient with Cvcell hyperplasia (case 2, Fig. 8). The values given inside the gland are MRC mU per gram determined by bioassay (S.E.:t: 15 to 18 per cent). The values given outside the gland are MRC mU per gram determined by radioimmunoassay. Nonshaded areas were taken for histologic study and immunocytochemical staining. Similar findings in three additional normal human thyroid glands have been made. From Wolfe et al. (1973),by permission.

response to hypercalcemia and abolition of the response to glucagon (Fig. 11). In that patient, the intravenous infusion of dibutyryl cyclic AMP failed to increase calcitoninsecretion. The relevance of these findings to the physiologic control of calcitonin-secretion in the human, or possibly to the pathogenesis of C-ceII hyperplasia and MCT remains speculative. It is possible, for instance, that a normal physiological interaction exists between the gastrointestinal hormones and the thyroid C-ceIls, protecting the individual against postprandial hypercalcemia. In patients with MCT, however, the ingestion of a

193

Medullary carcinoma of the thyroid

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FIG. II. Serum calcitonin (C'I) response to infused calcium and glucagon basally, and after aand /3-adrenergic blockade, in a patient with MCT. The range of untreated, resting values of serum CT is shown in the shaded bar at bottom of the basal infusion graphs. From Melvin et al. (1970a),by permission.

large oral load of calcium is a weak and inconsistent stimulus to CT-secretion (Melvin et al., 1972). Further studies are needed of the time-course of the CT-response to infused pentagastrin, and a more specific role for the gastrointestinal hormones as CTsecretagogues may yet be defined. MCT does not respond to thyroid stimulating hormone (TSH) as assessed by the serum CT response, and the tumor does not take up radioiodine (Melvin et al., 1972). Metabolic Effects of CT

Patients with CT-secreting MCT present a unique opportunity for observing the long-term effects of chronic endogenous CT-excess on calcium and bone metabolism. It would be expected that the net effect of CT must reflect not only the direct influence of the hormone on bone, but also the possible direct effect (Fischer et al., 1971; Deftos et al., 1972) and indirect effect of CT on the secretion of parathyroid hormone (PTH), and on Vitamin D metabolism (Olson et al., 1972) Although CT is hypocalcemic in effect, hypocalcemia occurs only very rarely in patients with MCT (for review, Melvin et al., 1972). It is possible that the parathyroid response prevents hypocalcemia in most cases, but not all patients with MCT have increased serum levels of PTH, and in one case (Melvin and Tashjian, 1968) severe hypocalcemia occurred in spite of high serum levels of immunoreactive PTH. Inhibition by CT of Vitamin D mediated calcium absorption has been observed in the rat (Olson et al., 1972) and this may be relevant to the rare instance of MCT and hypocalcemia. In patients with MCT, the increased secretion of CT does not appear to protect against an acute increase in serum calcium concentration (Melvin et al., 1973). In response to a standard intravenous infusion of calcium, no significant differences were noted between the magnitude and duration of hypercalcemia occurring in MCT patients, and in normal control subjects (Fig . 12). Conversely, however, prolonged JPTC Vol. I. No. til-/.!

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FIG. 12. Increment in serum calcium concentration in response to calcium infusion in twelve patients with familial medullary thyroid carcinoma (MCn ( - - ) , and twelve age-matched relatives with normal serum calc itonin (Cf') (- - -). Plotted are the mean values and standard error limits . During the infusion serum CT rose to high levels (> 2 ng/ml) in all patients with MCT but not in the norm al relatives. From Melvin et al. (1972), by permission.

hypercalcemia following calcium infusion has been noted in athyroidal subjects with presumed CT-deficiency (Hahnemann and Friis, 1965; Woodhouse and Barnes, 1968). The serum concentration of CT in patients with MCT is frequently far in excess of that achieved by therapeutic doses of exogenous CT currently being evaluated in the treatment of Paget's disease, and osteoporosis. The CT produced by MCT is known to be biologically active in experimental animals, and is identical in structure to the synthetic calcitonin-M which has a potent action in the human. Further, osteopetrosis has been described in association with C-cell tumors in bulls (Krook et al., 1969), and in four of nine children of a woman with MCT (Verdy et al., 1971). MCT provides us, therefore, with an experiment in nature perhaps anticipating the results of long-term studies presently being undertaken into possible beneficial effects of exogenous CT in the prevention or treatment of involutional osteoporosis. Data relating to quantitative assessment of bone mineral content in patients with MCT is presently very limited but the one study to date failed to demonstrate any differences between MCT patients and age/sex-matched normal control subjects (Melvin et al., 1973). In that study of eighteen patients with MCT and elevated basal serum CT levels ranging from 0.4-620 ng/rnl, bone density was assessed by means of photon beam absorptiometry of the mid-shaft of the radius (Cameron and Sorenson, 1963) and by radiological measurement of the metacarpal cortical index (Barnett and Nordin, 1960). Shown in Fig. 13 are the results of photon beam densitometry in ten female patients with MCT, ranging in age from 16 to 52 years. While the total duration of hypercalcitoninemia is unknown, high serum CT levels in association with metastatic MCT were known to be present in two patients for at least 2 and 3 years respectively. With two exceptions, the bone mineral content (g/cm2 ) was identical to the mean values obtained in normal age-matched controls. The two exceptions lay two standard deviations below the mean normal. In Fig. 14 are shown similar data for 8 male patients with MCT, ranging from 22 to 66 years of age. All had high basal serum CT levels ranging from 0.4 to 620 ng/ml. Two of these, aged 37 and 51 years, were known to have had metastatic MCT for at least 18 years, and in the younger of these, very high serum CT values (presently 620 ng/ml) were present when measured for the first time in 1967.

195

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40 SO AGE [YEARSI

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FIG. 13. Bone mineral content (gtcm') as measured by photon beam absorptiornetry of the mid-shaft of the radius in ten female subjects with high serum CT levels due to MCT. Three subjects had, in addition, high serum parathyroid hormone levels, and are annotated by circles (0). The normal range (mean ± 2S.0.) is that compiled by Cameron (1969) for over 500 normal female subjects. From Melvin et al. (1973),by permission.

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FIG. 14. Bone mineral content (gtcm') as measured by photon beam absorptiometry of the mid-shaft of the radius in eight male subjects with high serum CT levels due to MCT. Four subjects had, in addition, high serum parathyroid hormone levels, and are annotated by circles (0). The normal range (mean±2S.0.) is that compiled by Cameron (1969) for over 500 normal male subjects. From Melvin et al. (1973),by permission.

196

K. E. W.

MELVIN

In both these subjects, the bone mineral content was identical to the mean normal value for age-matched controls. In three of the eight patients, bone mineral content was less than normal. Measurements of the metacarpal cortical index in the same group of patients gave similar results to those obtained by photon beam densitometry, there being no differences between patients with MCT and normal control subjects. The oldest patient in the series, a male aged 66 years, had an abnormally low bone 'density as assessed by both methods. Much more data will be necessary before firm conclusions can be drawn, but there is presently no indication that in the human, chronic endogenous CT-excess has any net effect on bone density, either to increase bone density, or to protect against involutional osteoporosis. Suggestive evidence for a beneficial effect of administered CT in the treatment of osteoporosis has been reported in a number of short term studies (Milhaud et al., 1969; Hioco et al., 1969; Brown et al., 1970; Caniggia et al., 1970; Cohn et al., 1971). Conversely, detailed morphological studies (Jowsey et aI., 1971) have shown that in osteoporotic patients treated with porcine CT for up to 4 months, bone resorption rate increased rather than decreased, in association with an increase in immunoreactive PTH. Quantitative examination of bone histology in three patients with long-standing metastatic MCT and very high serum levels of CT (Melvin et al., 1973) indicated that in each case both bone resorption and bone formation had virtually ceased. The results shown in Table 2 indicate a striking decrease in the number and activity of osteoclasts, in the number of osteoblasts, and in the extent of osteocytic osteolysis. Based upon this limited evidence, the full therapeutic potential of exogenous CT will not be realized until some way can be devised for promoting bone formation while maintaining suppression of bone resorption. HISTAMINASE ACTIVITY

The enzyme histaminase is normally found in significant quantities only in human intestine, kidney, and placenta. Increased serum levels occur during pregnancy and following the injection of heparin. High tissue histaminase activity is found consistently in MCT and its metastases and appears to be virtually specific to MCT (Baylin et al., 1972). Elevated serum levels of the enzyme, however, do not occur regularly in patients with MCT, being detectable in 20 per cent of patients with localized disease, and 70 per cent of patients with metastatic disease. Highest serum levels have been found in association with pulmonary metastases. Figure 15 compares serum histaminase and serum CT levels as indicators of disease in sixteen patients with MCT, eleven of whom had overt metastases. Of the sixteen patients, normal basal values of serum CT occurred in four, and of histaminase in eight. The infusion of calcium resulted in abnormal CT levels in all patients, but did not influence serum histaminase levels. Thus, as a marker for MCT, histaminase lacks the sensitivity and consistency of CT. The physiologic significance of histaminase in the tumor is unknown. Administration of aminoguanidine, a specific inhibitor of histaminase, totally inhibited serum activity of the enzyme in all patients studied, but failed to modify serum levels of CT either basally or in response to calcium infusion. The drug does not appear, therefore, to affect overall tumor-cell function. DOPA DECARBOXYLASE

High dopa decarboxylase activity has been noted in each of five medullary carcinomas studied (Atkins et al., 1973), the levels being thirty to ninety times those found in adjacent normal thyroid tissue. It has been reported that C-cells in the thyroid of a variety of mammalian species contain dopa decarboxylase activity, an important enzyme in the synthesis of catecholamines (Hakanson et al., 1971).The finding provides further confirmation that MCT arises as a tumor of C-cells, and may be relevant to the mechanisms controlling calcitonin secretion. It has been postulated that monoamines

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FIG. 15. Corresponding basal serum calcitonin (Cf) and serum histaminase levels in sixteen patients with medullary thyroid carcinoma. Broken lines indicate, respectively, the maximum value of serum CT observed in 140 normal subjects, and 2 S.D. above the mean value for serum histaminase as measured in sixty-two normal subjects. Elevation of serum CT was evident in twelve patients, and of histamina se in eight. Of the four patients with marginal or normal serum CT values basally, all subsequently showed an abnormal rise in serum CT in response to calcium infusion. Note log scale used for serum CT. From Melvin et al. (1972),by permission.

play an important role in controlling the secretion of a variety of peptide hormones (Owman, 1973). If catecholamines were essential to the secretion of CT, it might be expected that MCT would contain also high tyrosine hydroxylase activity, the enzyme responsible for the important rate-limiting step in catecholamine synthesis. Although this enzyme was reported as being present in low concentration in two tumors (Atkins et al., 1973), this has not been the experience of others (Tashjian, A. H., Jr. and Wurtman, J., personal communication) who were unable to detect tyrosine hydroxylase in any of several medullary carcinomas tested. Further, in one patient with MCT, the intravenous administration of t-dopa, a substrate for dopa decarboxylase, resulted in an acute decrease in serum CT levels (Gagel, R., personal communication). Clearly, this aspect warrants further study. Of unquestioned significance is the fact that this demonstration of dopa decarboxylase in MCT further links the tumor to the APUD (Amine Precursor Uptake and Decarboxylation) cell system, the components of which take origin in the embryonic neural crest (Pearse, 1%9). The component cells of this system include thyroid C-cells, adrenal medullary cells, gastrointestinal chromaffin cells, pancreatic islet cells, and pituitary corticotrophs and melanotrophs. The concept has obvious relevance to the histogenesis of the MCT syndrome. ADRENOCORTICOTROPHIC HORMONE (ACTH) There are recorded at least seventeen cases of Cushing's syndrome occurring in association with MCT (for review, Melvin et al., 1970b). In several of these cases, ACTH or ACTH-like material was identified by means of biologic assay or immunoassay of the extracted tumor and patient's serum (Goldberg and McNeil, 1967; Donahower et al., 1%8; Szijji et al., 1%9; Vague et al., 1971). Shown in Fig. 16 are the

Medullary carcinoma of the thyroid

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FIG. 16. Plasma cortisol and ACTH values before and after excision of medulIary carcinoma of the thyroid in a patient with Cushing's syndrome. Dots annotated by circles represent values obtained while patient was receiving suppressive doses of Dexamethasone. Broken line indicates upper limit of normal for plasma ACTH. From Melvin et al. (I 970b), by permission.

data from a case of Cushing's syndrome caused by an ACTH-secreting MCT. Very high levels of plasma cortisol and immunoreactive ACTH fell to normal after thyroidectomy, accompanied by complete clinical remission of the Cushing's syndrome. The thyroid gland contained a unilateral medullary carcinoma in which there were large quantities of both ACTH, 26.5 mD/g, and CT, 39.1 MRC D/g. This author has experience with one other such patient, in whom the onset of Cushing's syndrome occurred 13 years after the diagnosis of MCT was made. PROSTAGLANDINS

High levels of prostaglandins E 2 and F 2a have been detected in the plasma of two patients suffering from MCT and in the tumor tissue of four (Williams et al., 1968).The authors believed that the levels present in the plasma were adequate to produce intestinal hypermotility and might in some patients account for the symptom of diarrhea frequently encountered in association with MCT. Others, however, have detected elevated serum levels of prostaglandins only infrequently, and in little or no relationship to diarrhea (Melvin et al., 1972; Tashjian, A. H., Jr. and Levine, L., unpublished). While the production of prostaglandins by MCT may explain the diarrhea in some patients, it does not do so in all. The diarrhea which occurs in this syndrome undoubtedly has a humoral basis, but the nature of the responsible circulating agent or agents remains to be elucidated. It is of interest that onepatient is reported to have found effective relief from the diarrhea of MCT by taking nutmeg, the active principle of which is myristicin (Fawell and Thompson, 1973). SEROTONIN

MCT may contain small amounts of 5-hydroxytryptamine (serotonin) in addition to certain monoamines (Ljungberg et al., 1967; Falck et al., 1%8; Ljungberg, 1972). The rare patient with MCT has shown an increased urinary excretion of 5-

200

K. E. W.

MELVIN

hydroxyindoleacetic acid (Moertel et al., 1965; Ibanez et al., 1967; Williams et al., 1968). Important humoral similarities between MCT and carcinoid tumors have been demonstrated, in which examples of both tumors secreted CT and prostaglandins (Kaplan et al., 1973).

HISTOGENESIS OF MCT-SYNDROME It has been suggested (Ljungberg et al., 1967; Schimke et al., 1%8; Weichert, 1970; Paloyan et al., 1970) that the medullary carcinoma syndrome can be attributed to a defect in a single cell system originating in the neural crest, and representing a familial chromaffinomatosis. A neural crest origin of the C-cells has been established (LeDouarin and Lievre, 1970). The neurochromaffin system, including the adrenal medulla, is known to be of neural crest origin. It is postulated that a common stem-cell in the neural crest gives rise also to cells which migrate into the primitive alimentary tract and form the enterochromaffin system, contributing also to the developing endocrine glands which bud off from the foregut. COllectively, these cells of neural crest origin constitute the APUD cell system, a terminology indicative of their shared capacity to take up and decarboxylate amine precursors. APUD cells share also a propensity for production of peptide hormones. C-cells and the C-cell tumor, MCT, produce the peptide hormone, CT, and contain both monoamines and the enzyme dopa decarboxylase, thereby qualifying for inclusion in the APUD cell series. Pheochromocytoma, an APUD cell tumor, may on occasion produce the peptide hormones CT and ACTH, and is rich in the enzyme dopa decarboxylase. Carcinoid tumors, arising from the enterochromaffin system, or the lung-itself a foregut derivative, may produce a variety of peptide hormones, including CT (Milhaud et al., 1972). Thus, on the basis of their common embryonic origin, the certain shared functional similarities and mode of inheritance, a link is reasonably established between the thyroid and adrenal components of the MCT syndrome. It seems likely that these abnormalities arise as a result of a single gene-defect in a common stem-cell in the neural crest. On this basis, also, it is not difficult to relate the other neuroectodermal elements of the syndrome. How the parathyroid component relates to this mechanism remains to be determined.

TREATMENT At present, the most effective treatment of MCT is surgical. Early diagnosis , i.e. detection of the carcinoma in situ by means of CT assay, makes surgical cure a reasonable expectation. Experience with radiotherapy in this condition is limited, but if CT secretion can be considered a measure of tumor-mass, radiotherapy proved ineffective in modifying the disease (Melvin et al., 1972). In view of the frequently multifocal and multiglandular nature of the disease, surgical management should include total thyroidectomy, identification and careful examination of all four parathyroid glands, and direct examination of both adrenal glands in the event of laboratory evidence of pheochromocytoma. Total thyroidectomy should be accompanied by total clearance of the lymph nodes of the central zone of the neck, and extension of this procedure to include clearance of the mediastinal lymph nodes through a sternal splitting procedure should frozen section reveal local node involvement (Miller et al. , 1972). When pheochromocytoma is present in addition, it is prudent that adrenalectomy be performed before thyroidectomy. This simplifies operative management, and permits direct examination of the liver for metastases. Hepatic metastases would make mediastinal exploration unjustified. The assay of CT in hepatic venous blood obtained by selective catheterization of the hepatic veins may also be useful in determining the extent of the surgical procedures. The role of chemotherapy in the treatment of MCT remains to be determined. In two patients, this author has seen no benefit from treatment with Bleomycin.

Medullary carcinoma of the thyroid

201

All relatives of known cases of MCT should be screened for familial disease by means of the calcium infusion test and serum CT assay (Melvin et al., 1971). CYTOGENETICS

Quinacrine mustard and Giemsa staining of peripheral leucocyte chromosomes in several patients with familial MCT has shown no abnormality of chromosome morphology or banding pattern (Melvin et al., unpublished). ONCOGENESIS

A 'two-hit' theory has been proposed in which the familial occurrence of MCT and pheochromocytoma is explained on the basis of a double mutation (Knudson and Strong, 1972). According to this hypothesis, the syndrome involves inheritance of one of the mutational events, probably through an autosomal dominant mechanism. In addition, a second mutational event in the post-zygotic period is required for the development of tumors. It is possible that viral invasion and transformation of genetically susceptible cells may cause the second, oncogenic, mutation. Such a mechanism has been suggested as implicating the Epstein-Barr (EB) virus in the oncogenesis of African Burkitt's lymphoma and nasopharyngeal carcinoma (Kafuko et al., 1972; Nonoyama and Pagano, 1973). In a kindred affected by MCT, significantly elevated titers of EB viral antibodies were detected in thirty-two family members, fifteen with MCT and seventeen without cancer (Li et al., 1974). Since increased serologic reactivity to EB virus occurred equally in cancer-patients and their unaffected relatives, the authors concluded that EB virus was not the causal agent of the tumor. They suggested, however, that the finding of increased serum reactivity to EB virus in members of that kindred might reflect an increased susceptibility generally to the oncogenic effects of other viruses. In the same study, cultured fibroblasts obtained from skin biopsies showed an increased transformation-rate by SV40 virus in four of sixteen family members, but again this phenomenon occurred both in those with and without the cancer. In all patients studied, no gross abnormality of immune mechanisms was detected, as assessed by quantitation of immunoglobulins, skin-testing with a variety of antigens, and stimulation of peripheral blood lymphocytes by phytohemagglutinin. CONCLUSION The MCT syndrome offers insights into genetic, embryonic, and pharmacologic interactions in the oncogenic process involving a variety of endocrine and nonendocrine tissues. Hormonal studies in affected patients have provided unique information concerning the mechanisms governing the secretion of calcitonin in the human and the possible role of that hormone in human physiology and metabolism. Recognition of the biosynthetic activities of the thyroid carcinoma and application of this knowledge to the serologic diagnosis of occult tumor has resulted in a completely reliable means of early detection, and improved expectations for treatment. ACKNOWLEDGMENTS Much of the original work included in this review was done in collaboration with Armen H. Tashjian, Jr., M.D., Professor of Pharmacology, Harvard School of Dental Medicine and Harvard Medical School; Harry H. Miller, M.D., Sergio Cervi-Skinner, M.D., Hubert Wolfe, M.D., Marilyn Melvin, M.S. Zoila Feldman. B.S., and the staff of the Clinical Study Unit, New England Medical Center Hospital, Boston, Mass. Grateful acknowledgment is made of their invaluable contributions. The investigations were supported in part by research grants (AM612, AM 10206) from the National Institute of Arthritis and Metabolic Diseases; General Clinical Research Centers Branch (FROO54); and American Cancer Society (CI-65).

202

K. E. W. MELVIN

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ADDENDUM Since completion of this manuscript, the intravenous administration of pentagastrin, 0.05Ilg/Kg body weight, has been demonstrated to be as effective as the calcium infusion test in the diagnosis of MCT or C-cell hyperplasia. The peak elevation of serum CT occurs 2 min after injection of Pentagastrin.