Routine serum calcitonin measurement in the evaluation of thyroid nodules

Best Practice & Research Clinical Endocrinology & Metabolism Vol. 22, No. 6, pp. 941–953, 2008 doi:10.1016/j.beem.2008.09.008 available online at http://www.sciencedirect.com

4 Routine serum calcitonin measurement in the evaluation of thyroid nodules Rossella Elisei *

Dr Department of Endocrinology, University of Pisa, Via Paradisa 2, Pisa, Italy

Calcitonin (CT) is secreted mainly by parafollicular C cells. In normal subjects, serum CT (sCT) levels are low and barely detectable by commonly used assays. Increased sCT levels are highly suggestive of medullary thyroid carcinoma (MTC). Since the clinical manifestation of MTC is a thyroid nodule, either single or in the context of a multinodular goitre, the routine measurement of sCT in the evaluation of thyroid nodule(s) facilitates the diagnosis of MTC. sCT measurement is indeed more sensitive than cytology in finding MTC. There are two major benefits from this clinical practice: (a) the surgeon is alerted to the need to perform total thyroidectomy and central compartment lymphadenectomy, which is the minimal surgical treatment for MTC; and (b) the outcome of MTC is favourably affected because it is usually identified at a less advanced stage. However, other non-MTC causes of hypercalcitoninaemia and false sCT positivity do exist and must be recognized. The differential diagnosis is possible using previously validated assays against possible sources of interference, and performing stimulation tests with pentagastrin or, eventually, calcium infusion. Key words: thyroid nodule; medullary thyroid carcinoma; calcitonin; pentagastrin test; calcium test.

PHYSIOLOGY OF CALCITONIN HORMONE Calcitonin (CT) is a 32-amino-acid small polypeptide hormone produced almost exclusively by parafollicular C cells.1,2 The gene encoding CT is located on chromosome 11p; through the mechanism of alternative splicing, this gene can generate two distinct mRNAs: CT and calcitonin-gene-related peptide (CGRP).3 In normal conditions, the two mRNAs are tissue-specific, as demonstrated by the almost exclusive finding of CT mRNA in the thyroid and of CGRP mRNA in the nervous system.4 CT is involved in calcium homeostasis, and its secretion is regulated mainly by extracellular calcium concentration.5 Other substances – such as pentagastrin,

* Tel.: þ39 050 995120; Fax: þ39 050 578772. E-mail address: [email protected] 1521-690X/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved.

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b-adrenergic agonists, growth hormone-releasing hormone and other gastrointestinal peptides – can stimulate CT release from parafollicular C cells.6–8 The physiological role of CT is still not well defined. It is known that CT specifically binds to the osteoclasts and that homozygous CT gene knock-out mice had a significant increase in bone formation, thus suggesting an inhibiting role of CT for bone resorption.9,10 However, in normal human subjects even quite large amounts of circulating CT have no major impact on serum Ca levels. Only in subjects with an increased bone turnover does exogenous CT administration acutely inhibit bone resorption and lower the serum Ca.11 Recently, evidence has been reported suggesting that the actions of CT may not be limited to bone. CT receptors have also been identified in the central nervous system, testes, skeletal muscle, lymphocytes, and placenta.12 CALCITONIN AS TUMOUR MARKER OF MEDULLARY THYROID CANCER Transformation of parafollicular C cells leads to the development of medullary thyroid carcinoma (MTC). This is a well-differentiated thyroid tumour which maintains the biochemical and pathological features of the C cells. Since malignant transformed C cells continue to produce and secrete large amounts of CT, elevated serum CT is strongly suggestive of the presence of either a primary MTC when the thyroid gland is still present, or metastatic MTC when the thyroid has been surgically removed.13 Only rare cases of de-differentiated MTC are associated to low or absent levels of serum CT.14 The prevalence of MTC varies from 5 to 10% among all thyroid tumours, and from 0.4 to 1.4% among thyroid nodules.15–18 Unlike other thyroid diseases, females and males are almost equally affected. The mean age at diagnosis is around 40 years, but a wide range of ages at onset has been reported.15,17–19 In about 25% of cases MTC is one of the components of the multiple endocrine neoplasia type-II syndromes, which are autosomal dominant inherited syndromes involving several endocrine glands (Figure 1). The pathogenetic mechanism of these syndromes has been recognized in the activation of the RET proto-oncogene.20,21 Several germline RET mutations, mainly concentrated in exons 10–16 of the RET gene, have been discovered to be associated with the hereditary form of MTC (Figure 2).22 Somatic RET mutations are also found in about 45% of sporadic MTCs and have been reported to have a poor prognosis both for outcome and survival (Figure 3).23–25 75% SPORADIC

• MTC 100%

MEN IIA: •

PHEOCHROMOCYTOMA 50%

• PARATHYROID MULTPLIE ADENOMATOSIS 30%

MTC

• MTC 100%

25% HEREDITARY

MEN IIB: • PHEOCHROMOCYTOMA 45% • • • •

(INCLUDED IN MULTIPLE ENDOCRINE NEOPLASIA SYNDROMES)

FMTC:

HABITUS MARPHANOID 65% MUCOSAL NEURINOMAS 100% MEGACOLON 30% CORNEAL NERVE HYPERTROPY 50%

• MTC 100% (ALONE)

Figure 1. Different forms of medullary thyroid cancer (MTC).

Serum calcitonin measurement in MTC 943

Figure 2. Most common RET-gene-activating mutations found associated with multiple endocrine neoplasia type II syndrome (MEN II) to date. RET mutations most frequently activate single-nucleotide point mutations, but some deletions and insertions have also been reported, especially at somatic level.

Since embryologically C cells originate from neural crest cells, MTC belongs to the neuroendocrine tumour group. This makes MTC distinct from the other well-differentiated thyroid carcinomas – i.e. papillary (PTC) and follicular (FTC) – which derive from follicular cells whose embryological origin is endoderm.26,27 The biological

MTC survival rate

100

No RET mutation

P = 0.006

75 RET mutation

50 0

5

10

15

20

25

30

Years of follow-up Figure 3. Patients affected with sporadic medullary thyroid carcinoma (MTC) harbouring a somatic RET mutation show a significantly lower rate of survival when compared with patients with sporadic MTC without a somatic RET mutation.

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behaviour of MTC, both sporadic and hereditary, is much less favourable when compared with that of the other well-differentiated thyroid carcinomas, even though it is not as unfavourable as that of anaplastic carcinoma.28 A 10-year survival of only 50% for MTC patients is reported in several series. The only possibility to improve the cure and survival of these patients consists in early diagnosis and early surgical treatment while the MTC is still intrathyroid.29,30 CALCITONIN MEASUREMENT IN THE WORK-UP OF PATIENTS WITH THYROID NODULES The most common clinical presentation of MTC is a thyroid nodule, either single or belonging to a multinodular goitre. No other specific symptoms or signs are present, with the exception of diarrhoea and/or flushing when an advanced metastatic disease is already present at the time of the diagnosis.29 The association of thyroid nodular disease with a lump in the neck may lead the clinician to suspect a thyroid malignancy, but not specifically an MTC. The most important diagnostic tool to identify thyroid malignancies is fine-needle aspiration cytology (FNAC).31 In a typical cytological smear of MTC, cells are usually isolated, varying in shape from oval to round, large polygonal or spindled. Cytoplasm may be abundant or scanty and usually contains acidophilic granulation visible with specific stains (May–Grunwald–Giemsa). There are usually two or more nuclei, typically round and eccentrically localized (Figure 4). Amyloid is frequently detectable as clumps of amorphous material, and is revealed by Congo red staining.32,33 The immunocytochemistry for CT and/or chromogranin (also secreted by MTC) should be performed if there is diagnostic uncertainty.34,35 Although the cytological pattern of MTC is generally typical, there are several series that show a high percentage of failures in making a cytological pre-surgical diagnosis.16,36–38

Figure 4. Binuclear cells, abundant cytoplasm and eccentric nucleus are the typical cytological features of medullary thyroid carcinoma (MTC). May–Grunwald–Giemsa stain, 100x.

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In 1991 we performed a pilot study measuring serum CT in all patients affected with thyroid nodule(s). We found that the sensitivity of serum CT measurement was higher than that of FNAC in detecting MTC.16 This finding was reproduced in several other European series.37–42 The prevalence of MTC varied from 0.57 to 1.37 according to different series, but in all cases serum CT sensitivity was demonstrated to be superior to the sensitivity of FNAC.16,37–39,43 Among other explanations, FNAC-negative results might be due to the MTC not being in a nodule submitted to FNAC, especially when multinodular goitre is the clinical diagnosis, or to a poorly defined cytology, which could require immunocytochemistry for CT not usually performed in a routine setting.44 It must also be considered that serum CT may be elevated even in the presence of microfoci of MTC not identifiable as nodules on neck ultrasound.16,39 Despite all these findings, the routine measurement of serum CT in patients with thyroid nodules is still a matter for discussion. While for the majority of European authors serum CT measurement should be included among the procedures to be performed in the presence of thyroid nodule(s)45, this is not the opinion of the American Thyroid Association guidelines task force; the latter did not find any level of evidence to recommend routine serum CT measurement in all patients with thyroid nodules.46 There are two major concerns to be overcome before obtaining an overall consensus on the routine measurement of serum CT: (a) the cost-effectiveness, and (b) the possibility of having elevated serum CT in the absence of an MTC (i.e. either hypercalcitoninaemia not due to MTC, or false-positive results of serum CT measurement). The cost–benefit problem MTC prognosis is strictly dependent on the stage of the disease at the time of diagnosis. It is very well known that MTC can be definitively cured only if still intrathyroid or in the presence of few lymph-nodes metastases.47 For this reason, only an early diagnosis can allow early surgical treatment that can definitively solve the problem and make MTC patients disease-free.30 In 2004 we published a study demonstrating that routine measurement of thyroid nodules not only allowed the identification of those cases of MTC that could be missed by FNAC, but also evidenced that a significant percentage of MTCs diagnosed with serum CT measurement were at stage I or stage II, while the prevalence of cases diagnosed with other techniques were at stage III and stage IV. As expected on the basis of the big difference in stage distribution, both the final outcome of the two groups of patients and the survival curve were significantly more favourable for the group diagnosed with the routine serum CT measurement (Figure 5).48 This experience has been confirmed by other European authors.49 Based on the evidence that routine serum CT measurement offers a greater opportunity to definitively cure MTC patients, the problem of the cost benefit should be automatically overcome. At present, the cost of serum CT assays ranges between US $35 and $100, depending on the country. Considering an average MTC incidence of 0.5% among thyroid nodules, the cost for detecting one MTC by routine CT measurement (at a mean cost of US $50) will be US $12,500. The same patient diagnosed and treated at a later stage, not achieving normalization of post-surgical serum CT, will undergo, at least annually for his/her entire life, a complex and expensive diagnostic work-up in search of residual disease and one or more additional surgical procedure (usually palliative) if, eventually, the site of the disease is localized. The cost of these additional tests and re-operations could be considerably higher than that paid for the detection of one single case at an early stage. Similar considerations have been

946 R. Elisei

% of MTC patients

1

P < 0.001

0,8

n = 25 Cured

n = 26 0,6

0,4

Persistent disease Deceased

n =16

n=8 0,2 n=2

n=1

0 CT SCREENED

OTHER DIAGNOSIS

Figure 5. Comparison of the outcome of patients with medullary thyroid carcinoma (MTC) diagnosed with serum calcitonin (CT) screening and MTC patients diagnosed at histology. MTC patients diagnosed with serum CT show a highly significantly better outcome, with a very small number of patients dying from the thyroid malignancy.

made by other authors when analyzing the cost benefit of the serum CT screening in other countries.16,50 A Canadian–American joint study has recently been published which confirms that routine measurement of serum CT in patients undergoing evaluation for thyroid nodules appears to be cost-effective, comparable to colonscopy and mammography screening, particularly in young patients with a long survival perspective.51 High levels of serum CT not related to MTC Hypercalcitoninaemia deriving from non-MTC diseases Elevated basal levels of serum CT are diagnostic of MTC. However, there are several other conditions, both physiological and pathological, in which basal levels of serum CT may be found to be elevated, and a differential diagnosis is needed, especially if the serum CT value is not very high. As shown in Table 1, we can distinguish two major groups of ‘non-MTC’ hypercalcitoninaemia: those unrelated52–55 and those related56–58 to thyroid diseases, the latter being usually associated with C-cell hyperplasia (CCH). An accurate medical history and physical examination can be useful in finding other reasons for hyper-production of serum CT. However, both the stimulation of CT secretion59 and the CT measurement in the wash-out of the needle used for the FNA60,61 may be of great diagnostic value in all cases with reasonable doubt. The false positives of serum calcitonin determination It is worth noting that CT precursors (pre- and procalcitonin) and peptides derived post-translation (katacalcin and N-terminal peptide) are also present in the blood of human subjects and may interfere with serum CT measurement. Artefactual recognition of larger CT precursors are commonly observed with one-site radioimmunoassay. This problem seems to be overcome by the most recent generation of CT two-site immunoradiometric assay (IRMA) which can specifically recognize the mature

Serum calcitonin measurement in MTC 947

Table 1. Hypercalcitoninaemia in pathological conditions other than medullary thyroid carcinoma (MTC).

(A) Unrelated to thyroid diseases Small-cell lung carcinoma Various neuroendocrine tumours Chronic renal failure Pernicious anaemia Zollinger’s syndrome Pancreatitis

(B) Correlated with thyroid diseases Lymphocytic thyroiditis Micropapillary thyroid carcinoma

calcitonin molecule.62 Artefacts may also be determined by the presence of heterophilic antibodies in the blood of patients that can interfere with the assay, thus producing false-positive results.63 Several manufacturers solved this rare and peculiar problem by directly adding mouse serum to the reagents of the assay. The mouse-serum antigens automatically bind the heterophilic antibodies and prevent their interference. Thus, the employment of a CT two-site IRMA method that has been demonstrated to have no cross-reaction with calcitonin precursors (i.e. pro-calcitonin) and to be protected against heterophilic antibody interference is highly recommended for routine serum CT measurement in thyroid nodule(s). THE CALCITONIN STIMULATION TESTS CT secretion is normally stimulated by several agents such as pentagastrin (Pg), calcium, alcohol and omeprazole.6–8 Subjects with elevated basal serum CT, without a clear cytological diagnosis of MTC, should be submitted to a Pg stimulation test (Table 2) before surgical treatment. The stimulation test allows CT secreted by an

Table 2. Stimulation tests with pentagastrin and calcium infusion.

(A) Pentagastrin stimulation test Commercial product: Pentagastrin (Cambridge Laboratories; Cambridge, UK) 500 mg/2 mL each ampoule Administration dose: 0.5 mg/kg Modality: rapid intravenous (in 1 min) Blood taken: basal, þ2 min, þ5 min, þ15 min Patient preparation: 6 hours fast

(B) Calcium stimulation test Product: calcium gluconate Administration dose: 2 mg/kg Modality: rapid intravenous (in 4 min) Blood taken: basal, þ5 min, þ10 min, þ20 min Patient preparation: 6 hours fast

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MTC to be distinguished from CT derived from other sources or due to technical artefacts.54,63,64 While a significant increase in serum CT is observed in patients with MTC (Figure 6A)59,65,66, the absence of a significant increase in serum CT levels after a Pg stimulation test (Figure 6B) strongly suggests either the artefactual nature of these false-positive values or their non-thyroidal origin (Table 1). A significant increase in serum CT after Pg stimulation may be observed in patients with thyroiditis or with small microPTC: both conditions can be associated with CCH that is responsible for the CT secretion. In this regard it is worth noting that it is rather difficult to distinguish ‘benign’ from ‘malignant’ CCH, and the pre-neoplastic role of CCH found in the absence of concomitant MTC foci is still open to debate.67 Several attempts have been made to find the ‘cut-off’ of the serum peak CT able to distinguish the presence of an MTC from CCH or other non-MTC hypercalcitoninaemias. According to some authors, a Pg stimulation test is considered as positive when the serum peak CT is at least 3 times the basal level, independently from the real absolute basal value.16 For others, a serum peak CT > 100 pg/mL must be considered as positive and strongly suggests the need to proceed to total thyroidectomy and central neck dissection.66 For serum peak CT between 60 and 100 pg/mL a grey zone is present, and the histological examination of the removed thyroid gland can fail to reveal MTC foci. Serum peak CT < 60 pg/mL is not diagnostic either for MTC or CCH, since also normal subjects may have an increase in serum CT after Pg stimulation up to 50–60 pg/mL.68 The mechanism by which Pg stimulates gastric acid, pepsin and intrinsic factor secretion is unknown; however, since Pg is an analogue of natural gastrin, it is believed that it stimulates the oxyntic cells of the stomach to secrete to their maximum capacity. In-vivo experimental studies have demonstrated that Pg increases blood flow in the gastric mucosa, inhibits absorption of water and electrolytes from the ileum, and promotes sodium and chloride diuresis. It causes contraction of the smooth muscle of the lower oesophageal sphincter when intravenously administrated.69–71 The Pg stimulation test has two major limits: unpleasant induced symptoms, and drug unavailability in several countries. Although side-effects are usually rare, when they do occur they may require medical attention. Patients can have skin rash or hives, nausea or vomiting, stomach pain, and urge to have bowel movement. Less common

B

200 175

Serum CT pg/mL

Serum CT pg/mL

A

150 125 100 75 50 25 0

200 175 150 125 100 75 50 25 0

0

2

5

15

Minutes after Pg injection

0

2

5

15

Minutes after Pg injection

Figure 6. Different patterns of calcitonin (CT) stimulation after pentagastrin (Pg) injection. (A) A typical positive response with a serum peak CT > 100 pg/mL, strongly suggestive of medullary thyroid carcinoma (MTC). (B) Negative response with a serum peak CT < 100 pg/mL, strongly suggestive of either a hypercalcitoninaemia not related to C-cell production or a false-positive CT determination.

Serum calcitonin measurement in MTC 949

but still possible are symptoms such as blurred vision; chills; dizziness, faintness, or light-headedness; drowsiness; fast heartbeat; feeling of heaviness of arms and legs; headache; increased sweating; numbness, tingling, pain, or weakness in hands or feet; shortness of breath; unusual tiredness; and unusual warmth or flushing of the skin.72 However, these side-effects usually have a very short duration (2–3 minutes) and can be safely managed at ambulatory level. As an alternative to Pg stimulation an intravenous infusion of calcium (Table 2) can also stimulate CT secretion when deriving from C cells (either CCH or MTC). This test is very well tolerated since there are no major side-effects. However, at present there are no controlled studies on the comparison of Pg and Ca stimulation tests, and the interpretation of the results of a Ca stimulation test is not as well defined as for the Pg test. Another fascinating old test, no longer employed in clinical practice, is the alcohol test.6 It is known that serum CT greatly increases after the administration of a small glass of whisky, but the test is not standardized and should be avoided. CALCITONIN MEASUREMENT IN THE WASH-OUT FLUID FROM FINE-NEEDLE ASPIRATION Although MTC can be diagnosed by FNA cytology (FNAC) based on typical pathological features32–35, the sensitivity of FNAC has been demonstrated to be only 63%, indicating that misdiagnosis often occurs with this approach.43 Recently, two studies have been published demonstrating that high CT concentrations were present in the wash-out of the needle used for FNA both in suspicious lymph nodes and in thyroid nodules histologically confirmed to be metastases or primary MTCs, respectively.60,61 The major concern in this approach is the cut-off to be used to define the positive value of CT in the FNA needle wash-out fluid. This problem is of course strictly dependent on the serum CT levels: when extremely high, contamination of the needle can occur and be misinterpreted as deriving from the nodule. However, only 5% of the serum CT concentration can be found as contaminant in the FNA needle wash-out fluid. An appropriate calculation taking into account the percentage of CT deriving from blood contamination allows the identification of the origin of CT measured in the FNA needle wash-out fluid.61 CONCLUSIONS In conclusion, routine measurement of serum CT in nodular thyroid diseases allows the preoperative diagnosis of unsuspected MTC and determines an early diagnosis of this relatively aggressive thyroid malignancy. The specific preoperative diagnosis of MTC is of great relevance in order to plan the appropriate surgical treatment that should be total thyroidectomy and central neck dissection on principle. The early diagnosis of MTC is also very important because, at present, an early diagnosis – when the primary tumour is still intrathyroid – can guarantee the success of the first surgical treatment and the definitive cure of MTC patients. The possibility of definitive cure in these patients is the main reason for which the cost benefit is effective. MTC patients not cured at first surgical treatment needed a long-term follow-up, with expensive annual controls accompanied by a lower quality of life, as compared patients successfully treated and declared disease-free.

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The technical problems and ‘non-MTC’ hypercalcitoninaemia may be easily overcome by using appropriate assays and the stimulation test. The CT measurement in the wash-out of the needle used for FNA can also be of great help in making the pre-surgical diagnosis of MTC.

Practice points  all patients with thyroid nodules should be screened for serum CT  elevated serum CT without a corresponding positive cytology should be tested with a stimulation test (pentagastrin or, eventually, calcium infusion)  serum peak CT after pentagastrin 3 times higher than the basal level or > 100 pg/mL is strongly suggestive for MTC  patients with serum peak CT between 60 and 100 pg/mL and any other evidence of MTC must be retested after 6 months  the CT measurement in the wash-out of the fine needle used for the cytological sampling may be useful  a two-site IRMA assay with no cross-reaction with CT precursors and protected against heterophilic antibodies should be used to avoid false-positive results.

Research agenda  a randomized study (CT-screened versus not CT-screened) including at least 4000 patients with thyroid nodules should be performed to verify the effectiveness of the early MTC diagnosis obtained with the routine serum CT measurement  a better definition of the positive CT response to calcium infusion  an autopsy study to verify the prevalence of micro-MTC in subjects deceased for reasons other than thyroid malignancy

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