Clinical performance of calcitonin and procalcitonin Elecsys^® immunoassays in patients with medullary thyroid carcinoma

Introduction

Medullary thyroid carcinoma (MTC) is a malignant tumor of the thyroid cells C cells, accounting for about 2–5% of thyroid cancers [1]. Calcitonin (CT), the main C cell secretory product, is considered a sensitive and specific marker for the diagnosis and follow-up of MTC [2]. However, CT assays suffer pre-analytic and analytic drawbacks: i. CT is prone to relatively rapid in vitro degradation by serum proteases which makes rapid processing of samples mandatory [3]; ii. the presence of various different immunoreactive isoforms and fragments, which can lead to inaccurate results (usually false low) as well as poor comparability of results obtained by different assays [4], [5], [6], [7]. Carcinoembryonic antigen (CEA), a nonspecific marker, is also used for monitoring MTC patients, being a reliable predictor of disease progression [8]; however, it is an insensitive marker for early postoperative detection of MTC persistence/relapse [9]. Administrating intravenous calcium or pentagastrin was largely used to improve the accuracy of CT measurement; however specificity is suboptimal and establishing clear reference guidelines for abnormal stimulated serum CT levels is a much more challenging task than establishing reference guidelines for abnormal basal CT levels. Accordingly, also considering additional costs and potential unpleasant side effects, most clinicians feel that provocative testing is no longer necessary [10]. Procalcitonin (PCT), the precursor of CT, circulates at low concentrations in healthy individuals; moreover, it has a concentration-independent and predictable in vivo half-life of 20–24 h and displays excellent in vitro stability in serum or plasma [11], [12]. A strong correlation between PCT and CT levels was proven in patients with MCT and multiple studies confirmed equivalent high sensitivity of CT and PCT in detecting and monitoring MTC [13], [14], [15]. On balance, the above-mentioned studies suggested that PCT, being less affected by analytical, physiological, pharmacological and pathological factors that can influence results of serum CT values, could be a new standard of care in the management of MTC [16], [17], [18], [19]. Accordingly, Lim and colleagues demonstrated a 98–99% negative predictive value for pathologically increased CT levels [20]. Recently, a new CT immunoassay become available on automated Cobas^® platforms (Roche Diagnostic, Rotkreuz, Switzerland) [21], [22] making feasible both simultaneous and sequential (reflex) measurement of CT and/or PCT on the same platform. Therefore, the present study was undertaken to compare the clinical performance of the automated Elecsys^® CT and PCT assays and their combination or sequential use for the diagnosis of MTC.

Materials and methods

This study was approved by the Ente Ospedaliero Cantonale Institutional Review Board and the Canton Tessin Ethical Committee, Bellinzona (Switzerland) [references CE 3466 and BASEC 2019-00662, respectively]. The study was performed in accordance with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects. A total of 226 subjects were included:

1. Patients with confirmed active MTC (n=16, 5 males, aged 31–78 yr; 11 females, aged 22–75 yr), based on clinical, imaging, or histological evidence of initial, persistent or recurrent disease. Patients with active MTC included six cases of newly diagnosed disease and 10 cases of metastatic disease.

2. Patients with confirmed MTC, deemed disease free by clinical assessment, imaging, and serum CT determinations (inactive MTC; n=23; five males, aged 22–73 yr; 18 females, aged 19–82 yr).

3. Patients with untreated benign thyroid diseases, including benign thyroid nodules, hyperthyroidism, hypothyroidism and goiter (n=125; 27 males, aged 26–75 yr; 98 females, aged 18–79 yr).

4. Patients with untreated (i.e. preoperative sampling) non-medullary differentiated thyroid cancer (DTC) (n=62; 19 males, ages 18–84 yr; 43 females, aged 20–76 yr).

The specimens were obtained from residual samples from patients of our Centre who had undergone CT and other thyroid testing at the EOLAB Central Laboratory (Bellinzona, Switzerland) between June 1st, 2016 and May 30th, 2018. Sera were stored at −80 °C immediately after clinical testing, until testing for Elecsys^® CT and PCT was performed for the present study. The final diagnosis and assessment of disease status was based on the longitudinal review of the clinical, imaging and biochemical data and any cytology/histology data available. Patients with renal failure were excluded because this may result in serum CT and PCT elevations.

Results

The distribution of serum CT and PCT concentrations in patients with active MTC, inactive MTC, benign and malignant non-MTC thyroid diseases is showed in Figure 1.

Figure 1:

Distribution of serum CT and PCT in patients with active MTC, inactive MTC, non-MTC benign and non-MTC malignant thyroid diseases, respectively [squares, median values; bars, interquartile ranges (IQR)].

Median serum concentrations of both markers were significantly higher in patients with active MTC compared with other groups. In addition, higher CT levels were found in patients with non-MTC benign disease compared to both inactive MTC and non-malignant MTC, respectively (Table 1).

No overlaps of PCT values were observed between patients with active MCT compared to other groups. Accordingly, the highest value in non-MTC patients (i.e. 0.20 ng/mL) was adopted as cutoff point to maximize the specificity without detriment in sensitivity. Applying the above described cutoff levels the corresponding sensitivity, specificity, positive (LHR+) and negative (LHR-) likelihood ratio values were 100%, 100%, 210 and 0 respectively. Discrepant results were found in five patients affected by non-MTC benign disease showing true-negative PCT but false-positive CT results, respectively (Table 2).

Since gender and thyroid volume is a relevant determinant for CT levels in non-thyroidectomized patients the performance of CT in discriminating patients with active MTC from those carrying benign and malignant non-MTC diseases was also evaluated by using i. the 97.5th percentile of CT distribution in males (3.7 pmol/L) and females (3.3 pmol/L) with non-MTC thyroid diseases (benign and malignant) and ii. the manufacturer-provided cutoff (males 2.8 pmol/L, females 1.9 pmol/L). Results are summarized in Table 3.

Discussion

This retrospective study evaluated the use of Elecsys^® CT and PCT as tumor markers in patients with MTC. First of all, no statistically significant difference was found between the two analytes in discriminating active MTC from MTC in complete remission. However, when the ROC curve-optimized cutoff used for the above purpose was adopted to discriminate active MTC from patients with non-MTC thyroid disease false-positive results were observed in five patients, affected by Graves’ disease (n=1) and multinodular goiter (n=4). This is not unexpected as CT levels increase in patients with enlarged thyroid glands; conversely these levels are low in patients with complete remission MTC due to previous surgery and absence of active disease. In addition gender-specific cutoff are suggested to improve accuracy. Accordingly, gender-specific cutoff levels obtained in our patients with non-malignant thyroid disease and those provided by the manufacturer and obtained in healthy subjects were also applied. No differences in sensitivity (100%) were observed using different cutoff levels (ranging from 2.8 to 3.7 pmol/L in males and 1.9–3.3 pmol/L in females, respectively). While manufacturer-provided cutoffs reduced specificity in both males and females those obtained in our patients with non-MTC thyroid diseases performed equally to ROC-derived one in males and slightly increased specificity and +LHR were observed in males and females as well. In summary, as the main results of our study i. both Elecsys^® CT and PCT assays were highly sensitive and specific MTC markers; ii. in the few cases with false-positive CT results a negative PCT measurement safely excluded active MTC. Putting our results in current clinical setting and considering that MTC management is centered on CT measurement since decades it seems reasonable, at the very least, to adopt PCT as complementary if not even as a replacement marker in patients with thyroid nodules and positive CT screening as well as in MTC patients with unclear serial CT measurement patterns after thyroidectomy. Furthermore, the present results, again confirming the value of PCT in MTC care, should contribute to long-term follow-up studies in MTC using PCT in order to further facilitate adoption of this novel, more reliable marker in MTC care. Having both assays performed on the same automated platform greatly facilitates the adoption of reflexing or reflective testing strategies avoiding inappropriate, even invasive, procedures to our patients with attached costs.

Figure 2:

Receiver operating characteristic (ROC) curve for calcitonin (sensitivity: active MTC, specificity, inactive MTC).