Combining serum calcitonin, carcinoembryonic antigen, and neuron‐specific enolase to predict lateral lymph node metastasis in medullary thyroid carcinoma

Abstract Background This study aimed to investigate the clinical application of combined detection of serum calcitonin (Ctn), carcinoembryonic antigen (CEA), and neuron‐specific enolase (NSE) in predicting lateral lymph node metastasis (LLNM) in medullary thyroid carcinoma (MTC). Methods Seventy‐four consecutive patients with MTC were enrolled. The relationship between serum Ctn, CEA, and NSE and LLNM was retrospectively analyzed by univariate analysis and logistic regression analysis. Furthermore, the clinical application of serum Ctn, CEA, and NSE combined detection in prediction of LLNM in MTC was also evaluated. Results The rate of LLNM in this study was 48.64% (36/74).The expression levels of serum Ctn, CEA, and NSE in MTC with LLNM were significantly higher than those without LLNM (all P < .01). The area under the curve (AUC) predicted by serum Ctn, CEA, and NSE for LLNM in MTC patients was 0.867, 0.831, and 0.726, respectively, and the AUC of serum Ctn, CEA, and NSE combined detection was up to 0.890, higher than using a single biomarker. The sensitivity and specificity of serum Ctn, CEA, and NSE combined detection in prediction of LLNM were 88.89% and 81.57%, respectively. Conclusions The concentrations of serum Ctn, CEA, and NSE are closely related to LLNM in MTC, and the combined detection of all three biomarkers has a higher clinical value in the evaluation of MTC patients with LLNM. With more perspective study in the future, it would be an indicator of influencing personalized surgical strategy for different MTC patients.


| INTRODUC TI ON
Medullary thyroid carcinoma (MTC) is a neuroendocrine neoplasm originating from thyroid parafollicular C cells and accounts for only 5% of all thyroid cancers, but its malignancy is relatively high, causing 8%-13% of thyroid cancer-related deaths due to its aggressiveness. 1 It has been reported that cervical lymph node metastasis is a common occurrence in MTC and more than 50% of patients with MTC have cervical lymph node metastasis at the time of initial diagnosis. Moreover, cervical lymph node metastasis plays an important role in evaluating the therapeutic effect and prognosis for MTC. 2 Therefore, the status of cervical lymph nodes in MTC patients should be carefully evaluated before treatment.Surgery is the only curative treatment for MTC. The treatment of cervical lymph nodes in current clinical practice includes the dissection of central and lateral cervical lymph nodes. Nowadays, central lymph node dissection is recommended for MTC patients according to the American Thyroid Association (ATA) guideline; however, it is still controversial whether lateral neck dissection is necessary for all the MTC patients. 3,4 Previous studies reported that MTC can synthesize and secrete a variety of bioactive substances, such as calcitonin (Ctn), carcinoembryonic antigen (CEA), and neuron-specific enolase (NSE). [5][6][7][8][9] Although numerous reports have shown that the expression levels of Ctn and CEA are related to cervical lymph node metastasis, the relationship between combined detection of serum markers and lateral lymph node metastasis (LLNM) in MTC patients is rarely studied.
In this study, we aim to analyze the differences of serum markers between MTC patients with LLNM (Group LLNM) and MTC patients without LLNM (Group non-LLNM), and to investigate the clinical application of combined detection of serum markers in predicting LLNM of MTC.

| Operation principle
Cervical lymph node dissection was performed during thyroidectomy. Principle of thyroidectomy: Patients with hereditary MTC, tumor size > 1 cm, extraglandular invasion, bilateral or multi-focus, and bilateral cervical lymph node metastasis were performed with total thyroidectomy, while patients with unilateral intrathyroidal tumors and tumor size ≤ 1 cm were treated with hemithyroidectomy and isthmectomy. Principle of cervical lymph node dissection: Central lymph nodes were routinely dissected, and lateral compartment (levels II-V) with evidence of lymph node metastasis should be dissection. The upper mediastinal lymph nodes (level VII) should be also cleaned if lymphadenopathy was present according to neck imaging. The recurrent laryngeal nerves should be directly visualized throughout the nodal dissection in order to avoid injury. The parathyroid gland and its blood supply should be retained.

| Detection of serum markers
Fasting blood samples were drawn from all patients before surgery in the morning and transported to our laboratory within 3 hours after phlebotomizing. After centrifuging at 3000 r/min for 10 minutes, serum was separated and analyzed. All specimens should be without jaundice, hemolysis and lipid. Serum Ctn was measured via Siemens IMMULITE ® 2000 automatic chemiluminescence immunoassay analyzer using a standard assay kit for in vitro diagnosis

| Pathological examination
The surgical specimens were diagnosed by two pathologists. The

| Statistical analysis
All the statistical analyses were performed using the SPSS package

| Comparison of serum markers in two groups of MTC patients
Patients with LLNM were classified as the Group LLNM, and the other patients were classified as the Group non-LLNM. Table 2 shows the relationship between expression levels of serum markers and LLNM in MTC patients. The expression levels of serum Ctn, CEA, and NSE in Group LLNM were significantly higher than the Group non-LLNM (all P < .01). However, there was no significant difference in the levels of serum AFP, CYFRA21-1, CA19-9, and CA242 between the two groups (all P > .05). Spearman analysis showed that the levels of serum Ctn, CEA, and NSE in patients with MTC were positively correlated with each other (r Ctn-CEA = 0.871, r Ctn-NSE = 0.407, r CEA-NSE = 0.465, all P < .001).

| Clinical value of serum Ctn, CEA, and NSE combined detection to predict LLNM in MTC patients
Univariate analysis above showed that serum Ctn, CEA, and NSE were related with LLNM in MTC patients. Thus, we derived the following prediction equation of serum markers combined detection for LLNM in MTC patients by logistic regression analysis (enter method) (as shown in Table 3

| D ISCUSS I ON
Cervical lymph node metastasis is an important factor of prognosis in MTC patients. Once the patients have lymph node metastasis in lateral compartment (level II to V), it should be classified as phase IV, which causes poor prognosis, and lateral neck lymph node dissection is then required. It has been reported that the rate of LLNM in MTC patients was 40%-66.7%. 10,11 This study found that the metastasis rate of lateral compartment was 48.64%, which was consistent with the previous literatures. Therefore, Iit is necessary to carefully evaluate the status of lateral lymph node in MTC patients before surgical operation. Ultrasound examination and computed tomography of the neck are recommended for patients with the extensive neck disease of MTC, 1 but their sensitivity is not ideal. 12 The purpose of this study was to investigate the clinical application value of serum mark- Ctn is a type of polypeptide hormone consisting of 32 amino acids which is released by thyroid parafollicular C cells with a molecular Ctn. 9,19 This study also showed that there was significant correlation between serum CEA and Ctn in MTC patients (rct-cea = 0.871). It was reported that the positive expression rate of CEA in MTC was also very high, and its concentration was closely related to the progression and prognosis of MTC, which has increasingly become an important indicator in the diagnosis and treatment of MTC. 5,8,9 A recent Canadian study of Canadian also reported that preoperative CEA level was closely related to tumor size, TNM stage, regional lymph node metastasis, biochemical cure, and survival rate. 9 The results of our study demonstrate that serum CEA in MTC patients with LLNM was significantly higher than that in the group without LLNM.

TA B L E 1 Clinical characteristics of patients
When serum CEA levels were beyond threshold levels of 29.68 ng/ mL, the AUC of predicting LLNM was up to 0.831, with sensitivity Based on the advantages of the above-mentioned serum markers, we attempted to establish a prediction model for LLNM of MTC using the logistic regression analysis. The results showed that the AUC of serum Ctn, CEA, and NSE combined to predict the LLNM of MTC was the highest (0.890), with a sensitivity of 88.89% and a specificity of 81.57%. Therefore, we recommend that these three markers can be combined to improve the accuracy of evaluating LLNM in MTC.
In conclusion, the expression levels of serum Ctn, CEA, and NSE are closely related to LLNM in MTC, and the combined de- However, there are still some limitations in this study. Due to the retrospective analysis, there may be an inevitable selection bias.
Prognosis analysis and a larger cohort study will be needed in the future to verify our findings.

O RCI D
Jun Zhang https://orcid.org/0000-0003-3313-6986 F I G U R E 1 Receiver operating characteristic curves of serum Ctn, CEA, and NSE combined detection for LLNM in MTC patients