Thyroglobulin Measurement in the Washout of Fine Needle Aspirates for the Diagnosis of Suspicious Cervical Lymph Nodes

Salvatore Sorrenti^1†, Salvatore Ulisse^1†, Cira Di Gioia², Valeria Ascoli², Daniela Bosco², Emma Rullo², Antonio Catania¹, Francesco Tartaglia¹, Giovanni Carbotta¹, Nicola Cornacchini³, Stefano Arcieri¹, Domenico Mascagni¹, Maria Ida Amabile¹, Laura Giacomelli¹, Alessio Metere¹, Daniele Pironi¹, Massimo Vergine¹, Massimo Monti¹ & Enke Baldini¹

¹Dept of Surgical Sciences, “Sapienza” Univ. of Rome, Italy
²Department of Radiological, Oncological and Anatomo-Pathological Sciences, “Sapienza” Univ. of Rome, Italy
³Dept of Surgery, S. Kliment Ohridski University, Sofia, Bulgaria

^† The first two authors provided an equal contribution.

*Correspondence to: Dr. Enke Baldini, Dept of Surgical Sciences, “Sapienza” Univ. of Rome, Italy.

Copyright © 2019 Dr. Enke Baldini, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abbreviations (if used)
FNAC - Ultrasound-Guided Fine-Needle Aspiration Cytology
Tgp - Thyroglobulin Protein
FNAB - Fine-Needle Aspiration Biopsies
CLN - Cervical Lymph Nodes
PTC - Papillary Thyroid Carcinomas
MTC - Medullary Thyroid Cancer
ATC - Anaplastic Thyroid Cancer
DTC - Differentiated Thyroid Carcinomas
PDTC - Poorly Differentiated Thyroid Carcinomas

Introduction
Thyroid cancer (TC) is the most common type of endocrine neoplasia, accounting for 3.1% of all new cancers and for 0.4% of cancer-related deaths worldwide in 2018 [1,2]. The majority of TC arise from the epithelial follicular cells, and are classified as differentiated thyroid carcinomas (DTC), comprising the papillary (PTC) and follicular (FTC) carcinomas, poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) [3]. Medullary thyroid carcinomas (MTC), originated from the parafollicular C cells, represent 5-10% of thyroid cancers with a 1-2% prevalence in thyroid nodules [3]. The annual incidence of TC has displayed an increasing trend in the last decades which, however, is not attributable to a real escalation in the onset of tumors, but rather to the improved detection of microcarcinomas in small non-palpable thyroid nodules [4]. The latter are very common, with a prevalence of 19% to 67% in the adult population, while malignant lesions occur only in about 5% of them [5,6]. Therefore, the first aim of clinical evaluation of thyroid nodules is to discriminate against their benign or malignant nature [5,6]. In this regard, fine-needle aspiration cytology (FNAC) represents the main diagnostic tool owing to its good diagnostic accuracy (84-95%), reproducibility and cost effectiveness [5-9]. Total thyroidectomy, with eventual dissection of the central and/or lateral compartment neck, is recommended for TC patients, followed in most cases by I131 ablation of the remnant [5,6,10,11].

Post-operative management of patients comprises periodic neck ultrasound, I131 whole-body scintigraphy, and serum Tg measurement after exogenous stimulation with recombinant human TSH 6-12 months after surgery [5,6]. DTC patients have a favorable prognosis, with 10-years-survival rate of roughly 90%. Nonetheless, approximately 10% to 15% of them exhibit persistent or recurrent TC, one third of whom face the loss of radioiodine avidity, poor response to conventional therapies and cancer-related death [5,6,10]. For such reasons, the identification of new prognostic molecular markers is highly required to improve the prognostic stratification of thyroid cancer patients [12-18].

Most of relapses appear within the central or lateral neck. When a mass in the thyroid bed and/or a lymphadenopathy are observed by ultrasound, FNAB is performed to diagnose a locally recurrent TC. Therefore, at present, FNAC is of crucial importance both in preoperative phase, to establish the surgical approach and prognostic stratification of TC patients, and in the follow-up to identify relapses [5,6,10,19- 22]. An important limitation of this technique is that it depends totally on pathologist’s experience for recognition of metastases from TC, metastases from extra-thyroidal neoplasms or non-tumoral diseases [23-25]. In addition, it is worth to note that inadequate cellularity prevents diagnosis in approximately 15% of cases [26-28]. Several studies suggested that measurement of thyroglobulin protein (Tgp) in the washout of the same needle used for FNAC could improve the diagnostic accuracy on CLN harboring metastases from DTC [29-46]. As a consequence, routine association of Tgp with FNAC in the diagnosis of CLN metastasis from DTC has been endorsed [29-46].

Here, we evaluated 45 CLN with histological report to assess the diagnostic value of Tgp, quantified in the washout of needles used for FNAC, in the identification of lymph nodal metastasis from thyroid tumors. The results showed that FNAC and Tgp have a similar diagnostic performance, and that Tgp could be informative in case of inadequate lymph node sampling.

Materials and Methods

Patients
From September 2004 to June 2016, FNAC and Tgp measurements were performed in parallel on suspicious CLN at the outpatients’ clinic of Endocrinology and Thyroid Diseases of the Polyclinic Umberto I of Rome (Italy). Forty-five CLN obtained from 36 consecutive patients, for which histological report was available, were selected for the present study. Of these 32 CLN, corresponding to 26 patients, were already analyzed in a previous report [44]. The present study received the approval of the ethical committee of the Polyclinic Umberto I (protocol no. 2615), and all patients signed the consent form. Twelve males and 24 females (median age 44 yr, range 20-76 yr) were subjected to FNAB in one or multiple suspicious CLN. Among them, 10 patients had been formerly thyroidectomized for PTC, and hence they were suspected of relapse (see table 1). All patients with preoperative diagnosis of TC underwent unilateral neck dissection. The histological analysis evidenced that, of the 45 CLN analyzed, 31 had metastatic PTC, 4 had metastatic MTC, 3 had metastatic ATC, and 5 harbored metastases from extrathyroidal cancers (metastasis from non- Hodgkin lymphoma were contained in 3 CLN, metastasis from a rhino-pharyngeal carcinoma in 1 CLN, and metastasis from lung carcinoma in 1 CLN). Cervical lymph nodes 41 and 45 (patients 32 and 36) of table 1, with a non-neoplastic cytological diagnosis underwent surgical removal because of the presence of thyroid nodule with cytological diagnosis of PTC, that following surgery was confirmed by histology.

Ultrasonography and Fine-Needle Aspiration Biopsy (FNAB)
Ultrasonography (US) was carried out by means of the Aplio XV (Toshiba, Japan) system equipped with a linear transducer (PLT-805AT). The following US parameters were considered suggestive of tumor spreading to CLN: rounded shape, long axis/short axis ratio < 1.5, irregular echotexture, absence of echogenic hilum, sharp borders, presence of microcalcifications, liquefaction or coagulation necrosis, aberrant distribution of vessels. Patients selected for FNAB on the basis of US characters were recommended not to take anticoagulants during the 5 days before procedure.

Each CLN was stung for aspiration two or three times with a 22-gauge needle, performing a minimum of 2 and a maximum of 4 passes every time for adequate sampling. The aspirates were expelled onto one or more glass slides and prepared as previously described [44]. After that, the needle was rinsed in 1 ml of sterile phosphate buffered saline (PBS), which was centrifuged at 1200rpm for 5min. in order to eliminate cellular debris. Supernatants were then transferred in new tubes and frozen at -20ºC until Tgp assay.

Cytological and Histological Analysis
Both slides and surgical samples were examined by three experienced pathologists (CDG, VA, ER and DB), which delivered consistent reports in all cases. All pathologists were unaware of Tgp results and, vice versa, the biologists who carried out the Tgp assay were uninformed of the pathologists’ diagnoses.

Tgp and Tg Autoantibody Measurements
All the FNAB washouts were analyzed within 10 days from collection. Tgp and Tg autoantibody assays were accomplished with, respectively, the immune-luminometric assay Tg-PluS and the Anti-Tgn kit from B.R.A.H.M.S. (Hennigsdorf, Germany). All samples were read in duplicate, and in some cases read back after dilution in PBS. Results were expressed as ng/FNAB, and Tgp values below 0.15ng/ml, i.e. the functional sensitivity of assay, were considered negative. All samples were negative for the presence of Tg autoantibodies.

Statistical Analysis
Performances of FNAC and Tgp tests were compared by the one-tail Fisher exact test and refereed as statistically significant when the pertaining p value was < 0.05. Statistical analyses were made using the Stata software, version 8.0 (College Station, Texas, Stata Corporation, 2003).

Results
As reported in table 1, FNAC turned out to be inadequate in 4 (8.9%) out of the 45 CLN samples analyzed. The FNAC diagnosis was correctly provided in 34 cases out of 41 CLN, with an overall accuracy of 82.9%. In the remaining 7 cases, cytology erroneously identified 4 metastatic PTC that histology reported as 2 metastatic MTC (table 1, CLN n. 19 and 20) and 2 metastatic ATC (table 1, CLN n. 12 and 13); of 3 cytological diagnoses of non-neoplastic CLN, 1 was found to be a metastatic PTC (table 1, CLN n. 31), and the other 2 were found to be lymphomas at histology (table 1, CLN n. 37 and 38). These 7 FNAC diagnoses were considered as false negative.

In agreement with other studies, we decided to adopt a Tgp cut-off of 1ng/FNAB, as it offered a good discrimination of tumors limiting false negative results at the same time [44, 46-48]. In these conditions, Tgp provided correct diagnoses in 41 CLN (31 true positive and 10 true negative), false negative results in 3 CLN, comprising 2 metastatic ATC (table 1, CLN n. 9 and 11), and one false positive in a CLN affected by lymphoma (table 1, CLN n. 28). Specificity, sensitivity, positive (PPV) and negative (NPV) predictive values, and accuracy are reported for FNAC and Tgp in table 2. It may be appreciated as Tgp showed a statistically significant higher NPV, while no differences were recorded among the two diagnostic approaches for sensitivity, specificity, PPV and overall accuracy. The comparison between these parameters of FNAC and Tgp was performed by analyzing either PTC and ATC taken together or the PTC cases alone (see table 3). In both situations, no statistically significant differences were observed among the different diagnostic results.

Discussions
In the present study, we showed that US-guided FNAC and Tgp assay had the same overall accuracy in the diagnosis of cervical lymph nodes (CLN) suspicious of malignancy. Although dependent on the experience and ability of the cytopathologists, FNAC was widely documented to be a reliable method for identifying CLN metastases, not only derived from thyroid cancers but also from other primary malignancies, as well as in recognizing non-tumoral diseases affecting CLN. Since the first observation, reported by Pacini and colleagues in 1992 (32), that Tgp determination in fine-needle washout could improve the accuracy of FNAC in the diagnosis of DTC metastatic CLN, several studies have confirmed its clinical utility, and its routine association with FNAC in the preoperative diagnosis of suspicious CLN has been recommended [11, 29-47].

Our experience on 45 CLN with available histological report encompassed cases of CLN harboring metastases from PTC, MTC, ATC, and non-thyroidal primary tumors. Considering the 36 CLN with metastases from PTC and ATC as a unique group, FNAC exhibited 90.6% accuracy, a datum in line with those reported from other studies [32,33,37,40,44,46]. In addition, Tgp evaluation on the same group offered a diagnostic performance comparable to that of FNAC. As expected, Tgp levels were very low in the three CLN harboring metastases from ATC, owing to the partial or total loss of expression of thyroid specific genes typically occurring in this cancer. In fact, in one of these lymph nodes the Tgp was still detectable and correctly indicated metastasis from thyroid carcinoma (CLN n. 13). Conversely, the absence or the low level (below the cut-off value of 1 ng) of Tgp in the other 2 lymph nodes were false negative results (CLN n. 10 and 12). The greatest diagnostic usefulness of Tgp was found on lymph nodes with inadequate FNAC (see CLN n. 1, 2 and 8), for which Tgp was able to provide the correct diagnosis. Similarly, in one case with cytological diagnosis reporting the absence of neoplastic features (CLN n. 31) Tgp correctly identified the metastatic nature of the CLN. False negative or non-diagnostic results characterize about 20% of FNAC diagnoses, and are frequently observed in presence of cystic or highly vascular lymph nodes [46]. It is in these conditions that Tgp determination could be of particular clinical utility, solving a major limit of the canonical FNAC due to the low cellularity of the collected samples. Actually, in order to reduce the cost bound to routine Tgp determination, we previously suggested that washout samples should be always collected, but analyzed for Tgp content only in case of uninformative or inconsistent FNAC diagnosis with respect to patient’s clinical parameters [44]. More recently, Zhang and colleagues adopted a standard procedure for the diagnosis of CLN in patients with previous or concurrent diagnosis of PTC [48]. They suggest to evaluate FNA cytology first, and to analyze Tgp content only in those CLN samples not diagnosed as metastatic PTC [48].

Conclusions
FNAC represents the standard modality of evaluation of lymph-nodal metastases originated either from thyroid cancers or from other primary tumors. In the specific case of DTC detection, it is possible to combine this technique with Tg protein measurement without the need for further sampling. The results of the present study confirm previous observations about the diagnostic utility of Tgp assay in the FNA washout of CLN suspicious of metastatic thyroid cancer, particularly in the case of inadequate lymph node sampling.

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