Measurement of Fine-Needle Aspiration Thyroglobulin Levels Increases the Detection of Metastatic Papillary Thyroid Carcinoma in Cystic Neck Lesions

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1 Measurement of Fine-Needle Aspiration Thyroglobulin Levels Increases the Detection of Metastatic Papillary Thyroid Carcinoma in Cystic Neck Lesions Brittany J. Holmes, MD; Lori J. Sokoll, PhD; and Qing Kay Li, MD, PhD BACKGROUND: Patients with previously resected papillary thyroid carcinoma (PTC) are monitored for disease recurrence/metastasis by ultrasound surveillance and fine-needle aspiration (FNA) cytology. However, accurate diagnosis in lesions with cystic degeneration may be difficult due to scant cellularity. In the current study, the authors evaluated thyroglobulin in FNA (Tg-FNA) for detecting metastatic and/or recurrent PTC in patients with cystic neck lesions after thyroidectomy. METHODS: The pathology records were retrospectively searched for patients with previously resected PTC and subsequent Tg-FNA on a cystic neck mass. Tg-FNA was measured in needle rinses using a Tg assay. The ultrasound findings, Tg-FNA concentrations, and cytological and follow-up histological diagnoses were correlated. RESULTS: A total of 21 FNA specimens of cystic lesions from 19 patients were identified. Of 7 cases with cytologic and subsequent histologic diagnoses of metastatic PTC, the median Tg-FNA level was 100,982 ng/ml. Of 8 cytologically benign cases, 7 cases had Tg-FNA levels < 0.2 ng/ml, and 1 aberrant case demonstrated elevated Tg-FNA of > 1000 ng/ml. For 6 cytologically equivocal cases, including 3 classified as atypical/suspicious for carcinoma, 2 classified as insufficient/acellular debris, and 1 classified as spindle cell neoplasm, 4 patients demonstrated markedly elevated Tg-FNA levels (> 150 ng/ml) with subsequent surgical confirmation of metastatic PTC, whereas 2 patients had Tg-FNA levels of < 0.2 ng/ml with negative follow-up. Using a cutoff value of 0.2 ng/ml, Tg-FNA demonstrated a sensitivity of 100% and specificity of 87.5%. CONCLUSIONS: Tg-FNA is a useful ancillary test that improves the detection of cystic PTC metastases. Particularly in cytologically nondiagnostic cases, the measurement of Tg-FNA helps to distinguish benign from malignant cystic lesions. Cancer (Cancer Cytopathol) 2014;122: VC 2014 American Cancer Society. KEY WORDS: thyroglobulin; fine-needle aspirate; cystic neck mass; metastatic papillary thyroid carcinoma. INTRODUCTION In patients with previously resected papillary thyroid carcinoma (PTC), monitoring for locoregional disease recurrence is critical to achieving the best long-term patient outcomes. 1 Annual serum thyroglobulin (Tg) levels are routinely used to monitor disease recurrence. 1 3 Although the sensitivity of serum Tg is increased when measured in conjunction with thyroxine withdrawal or recombinant human thyrotropin stimulation, serum Tg levels < 0.1 ng/ml are associated with a low risk of disease recurrence in patients who have undergone thyroidectomy and remnant ablation. 1 In addition, because cervical lymph nodes (LNs) are the most common site of disease recurrence, 2 guidelines recommend an annual neck ultrasound study for patients who are free of disease at their initial postoperative evaluation. 1,3 Features that suggest metastatic disease on ultrasonography include intranodal cystic change, Corresponding author: Qing Kay Li, MD PhD, Department of Pathology, The Johns Hopkins Bayview Medical Center, 4940 Eastern Ave, Building AA, Room 154B, Baltimore, MD 21224; Fax: (410) ; qli23@jhmi.edu Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland Presented in abstract form at the 61st Annual Scientific Meeting of the American Society of Cytopathology; November 8-12, 2013; Orlando, FL. Received: January 20, 2014; Accepted: January 22, 2014 Published online March 3, 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: /cncy.21413, wileyonlinelibrary.com Cancer Cytopathology July

2 microcalcifications, diffuse hyperechogenicity, a microlobulated margin, round shape, loss of echogenic hila, and mixed or central vascularity on color Doppler sonography. 4 If ultrasound examination of the neck identifies a suspicious LN, fine-needle aspiration (FNA) is necessary to evaluate the cellular morphology and confirm the diagnosis. 1 In general, FNA is highly specific and sensitive in diagnosing recurrent/metastatic PTC, particularly in patients with solid lesions. For a suspicious cystic LN, the most consistent ultrasonographic features include a thickened outer wall and internal nodularity or septations. 5 The lack of epithelium in cyst aspirates may lower the sensitivity compared with FNA of solid lesions, leading to a nondiagnostic interpretation. 6 8 In these cases, ancillary testing may provide useful information to guide clinical decision-making. Our previous study of 200 cases of cervical LN FNA specimens in patients with PTC who had undergone thyroidectomy demonstrated that measurement of Tg levels in FNA material (Tg-FNA) increases the detection of occult metastases. 9 Similar earlier studies have also demonstrated a role for Tg-FNA to aid in the diagnosis of disease recurrence/metastasis, 10,11 suggesting that it may be particularly helpful in diagnosing difficult cystic metastases. 12 From the data presented by Cignarelli et al on 6 cystic PTC metastases, 12 Tg-FNA demonstrated a sensitivity and specificity of 100% for the detection of PTC. Although these previous studies have suggested the usefulness of Tg-FNA in diagnosing suspicious cystic lesions, to the best of our knowledge, the sensitivity and specificity in this setting have not been well documented. In the current study, we pursued our prior observations (including previously identified and newly added cases of cystic lesions) to evaluate the usefulness of Tg- FNA for detecting cystic recurrent/metastatic PTC. We specifically examined the sensitivity and specificity of Tg- FNA in patients who had undergone thyroid resection and developed suspicious ultrasonographic cystic lesions. MATERIALS AND METHODS Case Selection After approval by the Institutional Review Board at The Johns Hopkins Medical Institutions, the medical records were retrospectively searched for patients with previously resected PTC and subsequent Tg-FNA over a 5-year period ( ); 15 cases were identified from January 2008 to April 2012 (which have been previously reported 9 ) and 6 cases were identified from May 2012 to January The following clinical and pathologic data were collected for each case: age, sex, date of original PTC diagnosis, date and results of FNA, Tg-FNA material, date and diagnosis of subsequent surgical resection specimen (if applicable), and clinical and radiographic followup (for benign cases). The cytologic and ultrasonographic findings, Tg-FNA levels, and histologic diagnoses were correlated. FNA Cytology Material was obtained by ultrasound-guided FNA performed by endocrinologists or interventional radiologists using a 25-gauge needle. Direct smears were prepared from needle aspirates, stained with the Diff-Quik method, and reviewed for immediate on-site evaluation. The wetfixed (alcohol-fixed) smears were also prepared and stained with the Papanicolaou method in the cytopathology laboratory. The final cytological diagnosis was made through the evaluation of both types of smears. Tg Measurement To measure Tg-FNA in the clinical chemistry laboratory, a dedicated FNA pass was performed without smears, and the FNA needle was rinsed with 1 ml of Hank balanced salt solution without heparin. Specimens were immediately transferred to the clinical laboratory and stored at 220 C for 0 to 4 days before thyroglobulin analysis. Tg concentrations were measured using an automated chemiluminescence immunoassay (Beckman Coulter Access/ Access 2 Immunoassay System; Beckman Coulter Inc, Brea, Calif) with an analytical sensitivity of 0.2 ng/ml. Statistical Analysis Receiver operating characteristic (ROC) analysis was performed using MedCalc software (version ; Med- Calc Software byba, Ostend, Belgium). For dot plots, undetectable Tg values of < 0.2 ng/ml were represented using values of 0.19 ng/ml. RESULTS A total of 21 FNA specimens of cystic lesions from 19 patients were identified. The patients ranged in age from 21 years to 73 years (mean, 46 years), and included 522 Cancer Cytopathology July 2014

3 Thyroglobulin-FNA of Cystic Neck Masses/Holmes et al 12 men and 7 women. Of the 19 patients, 18 had previously undergone either total thyroidectomy or lobectomy followed by completion thyroidectomy; the remaining patient underwent what was described as limited thyroid surgery without radioactive iodine ablation for PTC 40 years prior. Sixteen of the 18 patients treated with thyroidectomy received postoperative radioactive iodine ablation, 1 patient was temporarily lost to follow-up, and another patient had no discernible uptake on his postoperative whole-body iodine scan. The average time from initial PTC resection to follow-up FNA was 6 years (range, 0 to 40 years). For FNA sites determined by repeat cytology, ultrasound, and clinical impression to have no evidence of disease recurrence (10 sites), the average time from FNA to last follow-up was 16 months (range, 2 months-37 months). The results of Tg-FNA measurement are summarized in Table 1. In 15 cases, a definitive diagnosis of metastatic PTC (7 cases) or benign LN (8 cases) was established based on the cytomorphology (Fig. 1). For 7 cases with cytologic and subsequent histologic diagnoses of metastatic PTC (Figs. 1A and 1B), the smears revealed tumor cells arranged in loosely cohesive clusters or flat sheets. Nuclei had fine granular or pale chromatin with nuclear grooves, pseudointranuclear inclusions, and small or inconspicuous nucleoli. Among these cases, the median TABLE 1. Thyroglobulin Levels in FNA Specimens of Cystic Neck Masses Cytologic Diagnosis Median Thyroglobulin, ng/ml Clinicopathologic Follow-Up Metastatic PTC (n57) 100,982 (range, ,600) Metastatic PTC (n57) Benign (n58) <0.2 (except 1 case at 13,606) Benign (n58) Other (n56) <0.2 Benign (n52) Atypical/suspicious (n53) 838 (range, ) Metastatic PTC (n54) Non-dx/insufficient (n52) Spindle cell lesion (n51) Abbreviations: FNA, fine-needle aspiration; Non-dx, nondiagnostic; PTC, papillary thyroid carcinoma. Figure 1. (A and B) Cytomorphology of recurrent/metastatic papillary thyroid carcinoma is shown. The smears revealed clusters of or scattered individual epithelioid cells with enlarged nuclei, vesicular chromatin, nuclear grooves, and pseudointranuclear inclusions. (C and D) Cytomorphology of benign cystic degeneration is shown (A: Diff-Quik stain, 3 400; B: Papanicolaou stain, 3 400; C: Diff-Quik stain, 3 100; D: Papanicolaou stain, 3 200). Cancer Cytopathology July

4 Figure 2. Thyroglobulin levels from fine-needle aspiration (FNA) material in cases diagnosed cytologically as benign (8 cases) or malignant (7 cases) with subsequent clinicoradiologic or histopathologic confirmation are shown. PTC indicates papillary thyroid carcinoma. Tg-FNA level was 100,982 ng/ml (range, 3.4 ng/ml- 956,600 ng/ml) (Fig. 2). Of 8 cytologically benign cases (Figs. 1C and 1D), 7 cases had Tg-FNA levels < 0.2 ng/ml, and in 1 case the Tg-FNA was elevated to > 1000 ng/ml (Fig. 2). This aberrant case demonstrated benign polymorphic lymphocytes on cytology and was described radiologically as a stable, unchanged LN for 3 years after the FNA. However, the patient s serum Tg was detectable up to 2 ng/ml while the patient was receiving thyroid hormone suppression and 5 ng/ml after recombinant human thyrotropin stimulation, raising suspicion of metastatic PTC that may have been missed due to sampling error on the FNA pass submitted for smears. The patient opted for conservative observation and declined surgical intervention. A ROC analysis comparing the benign and malignant groups revealed an area under the curve of Using a cutoff value of 0.2 ng/ml, Tg-FNA was found to demonstrate an overall sensitivity of 100%, a specificity of 87.5%, an apparent positive predictive value of 87.5%, and an apparent negative predictive value of 100% for distinguishing nonmetastatic from metastatic cystic lesions. Six cytologically equivocal cases included 3 classified as atypical/suspicious for carcinoma, 2 classified as insufficient/acellular debris, and 1 classified as a spindle cell neoplasm diagnosed by cytology (Fig. 3). The smears were usually scant, with a few atypical cells demonstrating round to oval nuclei, granular chromatin, and occasional nuclear grooves. No pseudointranuclear inclusions were Figure 3. Cytomorphology of fine-needle aspiration material in equivocal cases is shown. The smears were usually scant with few atypical cells demonstrating round to oval nuclei, granular chromatin, and occasional nuclear grooves but lacking pseudointranuclear inclusions (A: Diff-Quik stain, 3 400; B: Papanicolaou stain, 3 400). identified. Of these cases, 4 patients demonstrated markedly elevated Tg-FNA levels > 150 ng/ml (median, 838 ng/ml [range, 179 ng/ml-9225 ng/ml]) (Fig. 4) with subsequent surgical confirmation of metastatic PTC. Two patients had Tg-FNA levels of < 0.2 ng/ml with negative follow-up by repeat FNA (1 patient) and imaging (1 patient). Based on the cutoff value of 0.2 ng/ml derived from the ROC analysis, FNA Tg levels were found to have correctly classified the cytologically equivocal or nondiagnostic cases. DISCUSSION In general, a cystic neck mass raises a broad differential, including branchial cleft cyst, salivary gland neoplasm, epidermal inclusion cyst, metastatic squamous cell 524 Cancer Cytopathology July 2014

5 Thyroglobulin-FNA of Cystic Neck Masses/Holmes et al Figure 4. Thyroglobulin levels from fine-needle aspiration (FNA) material in cytologically equivocal cases diagnosed as atypical, insufficient, or spindle cell neoplasm (6 cases) are shown. The cases were subsequently classified as benign or malignant based on clinicoradiologic or histopathologic follow-up. PTC indicates papillary thyroid carcinoma. carcinoma, and metastatic PTC. 7 Cytologic examination of FNA material from cystic neck masses may be limited by a lack of epithelial cells for review. 6,7 Complete cystic degeneration of LNs due to metastatic PTC is most common in young patients aged < 35 years. 5 Thus, judicious use of cost-efficient ancillary testing concurrent with FNA of a cystic neck mass may increase both the yield and diagnostic accuracy of FNA. In the current study of cystic lesions, 28.5% of cases (6 of 21 cases) yielded a cytologically equivocal or nondiagnostic result. This is similar to the rate reported in previous studies, 10 and reflects the challenging nature of diagnosing cystic cervical LNs on FNA. Although some authors have found equivalent diagnostic yield and accuracy between cystic and solid FNAs of the head and neck, 13 others have reported that cystic lesions yield less diagnostic material, leading to lower sensitivity. 6 8 The results of the current study demonstrate that in cases with ambiguous cytologic features or inadequate material for evaluation, Tg increases the sensitivity and specificity of the FNA procedure. As the analytical sensitivity of immunologic assays for thyroglobulin has increased, the threshold of detection has decreased. Previous studies of combined solid and cystic lesions in patients both before and after thyroidectomy used the mean Tg-FNA level in patients without metastatic PTC plus 2 standard deviations as the cutoff value for malignancy. 12,14 However, a lower threshold is more sensitive in patients who have undergone thyroidectomy. In the current study, Tg was found to perform well at a cutoff value of 0.2 ng/ml for cystic lesions, and correctly classified 20 of 21 samples as benign or metastatic PTC. Our previous study of both solid and cystic lesions demonstrated a sensitivity of 97% and a specificity of 81% using a cutoff value at the Tg detection limit (< 0.2 ng/ml); comparison with the results of the current study indicates that the diagnostic thresholds for solid and cystic lesions are similar. Other studies of cervical FNAs, including both solid and cystic lesions, have also used thresholds near the level of detection of the assay (0.2 ng/ml-0.9 ng/ ml) in patients who have undergone thyroidectomy. Reported optimized cutoff values are 1.8 ng/ml to 1.9 ng/ml, ng/ml, 16 and 0.2 ng/ml to 0.7 ng/ml. 10 It is interesting to note that Tg may be undetectable in metastases of poorly differentiated PTC or anaplastic thyroid carcinoma, necessitating careful cytologic review independent of ancillary test results. 17,18 To maximize the benefit of ancillary testing in the preoperative setting for primary diagnosis, Chung et al proposed using Tg-FNA only in LNs with 1 or 2 suspicious features on ultrasound, not in LNs with multiple suspicious findings. 19 Suspicious features found to be most predictive of benefit from Tg measurement were cystic changes, hyperechogenicity, calcifications, and peripheral vascularity. 19 Applying a similar approach to monitoring for recurrent disease would triage specimens for FNA alone versus Tg-FNA, thereby avoiding excess testing in cases that are likely to yield diagnostic results. Similarly, Baldini et al suggested collecting a sample for Tg analysis from all FNA specimens, but only performing the testing on cases with inadequate cytology or inconsistent results based on the clinical impression. 18 However, to the best of our knowledge, the selection of cases for Tg- FNA has not been studied extensively in the postthyroidectomy setting. Antithyroglobulin autoantibodies (TgAb) are known to interfere with immunometric assays for serum Tg. 1,2 In multiple studies, TgAb have shown no effect on the sensitivity of Tg measurement in FNA needle washout. 10,17,20 This suggests that a separate measurement of TgAb may not be necessary for an accurate assessment of FNA material. However, in rare cases, high-titer TgAb in the Tg-FNA fluid may decrease the measured Tg level below the diagnostic threshold, leading to a false-negative result. 21 Although some laboratories have chosen not to Cancer Cytopathology July

6 measure TgAb routinely, 19 others measure either serum or FNA fluid values to flag rare patients with high-titer TgAb who are at risk for spuriously low Tg levels in Tg- FNA. 18,21 Finally, in the current study, we also found 1 case that demonstrated benign polymorphic lymphocytes on cytology and was radiologically stable 3 years after the FNA was performed. However, the patient s elevated Tg- FNA and detectable serum Tg were consistent with metastatic PTC, raising concern for sampling error. Thus, a further large-scale study of the potential effects of serum Tg levels on Tg-FNA is necessary. The results of the current study demonstrate that Tg measurement in FNA material appears to be a useful ancillary test that improves the detection of cystic PTC metastases. Particularly in cytologically nondiagnostic cases, the measurement of Tg-FNA helps to distinguish benign from malignant cystic lesions. Preliminary research has suggested that the detection of Tg mrna in FNA material further increases the sensitivity and specificity when added to Tg-FNA. 18 Future studies will elucidate whether the combination of molecular studies with Tg-FNA optimizes test performance. FUNDING SUPPORT Partially supported by the Drs. Ji and Li Family Cancer Research Grant (to Dr. Li). CONFLICT OF INTEREST DISCLOSURES The authors made no disclosures. REFERENCES 1. American Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid Cancer, Cooper DS, Doherty GM, et al. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid. 2009;19: Tuttle RM, Ball DW, Byrd D, et al; National Comprehensive Cancer Network. Thyroid carcinoma. J Natl Compr Canc Netw. 2010;8: Pacini F, Castagna MG, Brilli L, Pentheroudakis G, ESMO Guidelines Working Group. 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Serum antithyroglobulin antibodies interfere with thyroglobulin detection in fine-needle aspirates of metastatic neck nodes in papillary thyroid carcinoma. J Clin Endocrinol Metab. 2013;98: Cancer Cytopathology July 2014