Colloid and pigmented histiocytes in lymph node aspirates as a clue to metastasis in patients with a history of papillary thyroid carcinoma

Cystic changes, calcification, colloid material, and multinucleated giant cells are frequently associated with primary and metastatic papillary thyroid carcinoma (PTC). These features are sometimes present in negative lymph node fine‐needle aspiration cytology (FNAC). This study aims to review nodal aspirates of PTC to elucidate the significance of these cytological features in aspirates without tumor cells.

2][3] These features can be observed in lymph node fine-needle aspiration cytology (FNAC) specimens of PTC patients, even in cytologically negative aspirates without tumor cells seen.False negativity is a significant issue in lymph node aspirates. 4In this study, nodal aspirates with histologic follow-up from patients with PTC were reviewed for cytological features Background lymphoid and inflammatory component-presence of lymphoid fragments, lymphocytes, polymorphs (Figure 1).
Definitions and methods of assessment are detailed in Table 1.

| RESULTS
A total of 113 aspirates from 74 patients were retrieved.There were 25 male and 49 female patients, with an average age of 54.03 years, ranging from 20 to 95 years old.Of the 113 aspirates, there were 101 specimens with liquid-based preparations, 34 with smears and 74 with cell block preparations.Ninety-five out of the 113 (84.1%) aspirates were confirmed histologically with metastatic PTC.The majority of aspirates were from the left (n = 62/113, 54.9%) and right neck (n = 47/113, 41.6%), with two specimens from the axilla (1.8%) and supraclavicular fossa (1.8%) (Table 2).
Tumor cells were present in 79 cases, of which all were confirmed histologically.In the remaining 34 cytologically negative aspirates (i.e., aspirates without PTC tumor cells), 16 were positive for metastatic PTC on histology (false-negative) and 18 were true negatives with a corresponding negative histology (Table 3).on multivariate analysis (Table 3).

Comparing cytomorphological features of aspirates with metastatic
T A B L E 1 Definitions of cytologic parameters assessed.with lymphoid fragments being associated with a negative node on histology, while colloid material and pigmented histiocytes were associated with positive node status (Table 4).

| DISCUSSION
2][3] Histologically, PTC with cystic changes are easily recognized by an empty space containing fluid material, debris, blood, pigmented and foamy histiocytes among other inflammatory cells, calcification, and tumor cells. 1 Contents of the cyst are variably sampled in FNACs, 5 and the presence of cyst content, in particular a composition rich in histiocytes and acellular material, is often regarded as diagnostic of a cystic lesion. 6In FNACs, lining epithelial cells (tumor cells in the case of cystic PTC) are not consistently sampled in FNAC specimens, duct obstruction and hemorrhagic, degenerative and pseudocystic lesions can display highly similar morphological features but lack epithelial cyst lining. 7,8With the broad differential diagnoses including benign and malignant cystic lesions in the head and neck, the sole presence of cystic changes in an aspirate is not specific. 6wever, in the context of a patient with PTC, the presence of cystic changes in lymph node aspirates holds greater clinical significance.Apparent negative aspirates of cystic nodal metastasis of PTC only displaying histiocytes but not tumor cells have been reported. 9,10ereas benign cystic lesions are not infrequent in the head and neck organs such as the thyroid and the salivary glands, It should be noted that cystic changes in lymph nodes more commonly heralds metastatic malignant neoplasms (i.e., benign cystic change/degeneration is rare in lymph nodes). 11,12The history of PTC with the sampling anatomical location of a lymph node drastically increases the pre-test probability for cystic metastases, thus cytologic evidence of cystic changes should not be taken lightly.Of note, efforts of reducing false-negative diagnosis by application of immunocytochemical and biochemical markers, notably CK 19 and thyroglobulin in cyst fluid have been described. 13,14ese markers have potential in improving detection of metastasis but are limited to samples with fresh cyst fluid or cell block preparations.
In this cohort, foamy and pigmented histiocytes were associated with a positive histologic follow-up (Table 3).In subgroup analysis of aspirates without tumor cells, pigmented histiocytes independently associated with the presence of metastatic PTC on histology (Table 4).
In most cases, pigmented histiocytes were present in abundance and were readily recognizable at low power (Figure 2C).However, other cytological features of cystic changes, namely debris and extracellular pigment, were not specific for metastatic PTC.
PTC tumor cells not only display the classical nuclear features with grooves and inclusions, 15 but also are often accompanied by calcification, multinucleated giant cells, and colloid material. 16These features can be seen regardless of cystic changes, and are considered as a sign for possible PTC in thyroid aspirates. 17These features were all associated with positive histology in lymph node aspirates of the cohort.In the presence of calcification, multinucleated giant cells and colloid material are highly correlated with tumor cells, and not independently predictive of nodal metastasis.But for apparently negative aspirates, calcification and colloid material were specific for metastatic PTC, with all cases displaying these features being positive on histologic follow-up (Figure 2B).Colloid material was also independently associated with metastasis in those aspirates on multivariable analysis (Table 4).
Biopsy of lymph nodes suffers from limitations in sampling.Falsenegative rates of up to 30% have been reported in image-guided core biopsies, 18,19 with the main reason being failure in sampling nodal tissue and/or metastatic disease. 18The false-negative rate for imageguided lymph node aspiration cytology is higher, 20 and is further increased when the FNAC procedure is performed free-handedly. 4 A rich lymphoid component, including the presence of lymphoid fragments, germinal center tissue, and abundant dispersed inflammatory cells in the background have been proposed as cytologic criteria for lymph node adequacy. 21This cohort demonstrated correlation between dispersed lymphocytes and the presence of lymphoid fragments and negative histology in the corresponding lymph node.The presence of lymphoid fragments was also independently associated with a negative follow up in overall analysis and subgroup analysis of cytologically negative aspirates only (Tables 3 and 4).The falsenegative rate of negative aspirates was 72.2% (n = 13/18) for cases without lymphoid fragments, as opposed to 18.8% (n = 3/16) in those with lymphoid fragments identified.
The findings of this study showed that when tumor cells are absent in an aspirate, cytological features of cystic changes (necrotic material, pigmented histiocytes) and colloid material indicate a high risk of false negativity, whereas identifying lymphoid fragments increases the negative predictive value of lymph node aspirates.It has to be noted that only cases of PTC proven by thyroidectomy were recruited.Not all malignant neoplasms of the head and neck are associated with cystic metastasis, and colloid production is only limited to differentiated thyroid carcinomas. 15,22As most negative diagnoses are not followed by further surgical management, the number of such cases excluded due to lack of histologic confirmation was greater than aspirates positive for malignancy.In view of the limited sample size, a larger study would better confirm these findings.

| CONCLUSION
Other than the presence of tumor cells with the characteristic nuclear features of PTC, lymph node metastasis of PTC is associated with unique cytological features including cystic changes (foamy and pigmented histiocytes), calcification, colloid material, and multinucleated giant cells, which when identified on lymph node aspirates, are highly suggestive of tumor metastasis.Pigmented histiocytes and colloid material are independently associated with nodal metastasis, and when seen in an apparently negative aspirate without tumor cells, should raise suspicion in patients with a history of PTC.Calcification and/or multinucleated giant cells were only significant on univariable analysis and may be reported as atypical findings.On the contrary, the lymphoid fragments are evidence of adequate sampling and correlate with a true negative result.These three features should be searched for in negative aspirates to guide further investigation and/or management of the lymph nodes.

1 FF
Background lymphoid and inflammatory component.(a) A large lymphoid fragment, H&E, Â200 magnification.(b) Lymphoid fragments among a background of abundant lymphocytes, pap stain, Â200 magnification.[Color figure can be viewed at wileyonlinelibrary.com]I G U R E 2 Cytological features associated with papillary thyroid carcinoma (PTC).(a) Calcification, H&E, Â200 magnification.(b) Colloid material an aspirate without tumor cells, pap stain, Â200 magnification.(c) Multinucleated giant cell, H&E, Â400 magnification.(d) Clusters of metastatic PTC cells, H&E, Â400 magnification.(e) Multinucleated giant cells and pigmented histiocytes in corresponding thyroidectomy sections, H&E, Â400 magnification.(f) Psammomatous calcification in a lymph node with metastatic papillary carcinoma, H&E, Â400 magnification.[Color figure can be viewed at wileyonlinelibrary.com] associated with cystic changes, tumor associated features, and lymphoid components.Findings from this study will elucidate the significance of these cytological features in aspirates without tumor cells. 2 | METHODOLOGY A computerized search was performed for cases of PTC in the institute from the year 2001 to 2022 confirmed by thyroidectomy.All pathology reports (including surgical pathology and cytology) of each patient yielded from the search were reviewed to identify neck dissections and lymph node biopsies, with matched lymph node FNACs within 1 year prior.Cases with chemotherapy or radiotherapy during the interval between FNAC and histologic confirmation were excluded.The slides of the FNAC specimen, including all liquidbased, smear, and cellblock preparations, were reviewed by two board-certified pathologists, who were also blinded to the histologic follow-up.All discrepancies were resolved by viewing the slides together until a consensus was reached.Cytologic parameters reviewed include: I G U R E 3 Cytological features associated with cystic change.(a) Debris with adjacent tumor cell cluster, pap stain, Â200 magnification.(b) Isolated foamy histiocytes with multivacuolated cytoplasm, H&E, Â400 magnification.(c) Necrotic material, pap stain, Â400 magnification.(d) Scattered pigments and pigmented histiocytes, pap stain, Â200 magnification.[Color figure can be viewed at wileyonlinelibrary.com] PTC confirmed by histology to aspirates of negative nodes, presence of calcification (p = .044),colloid material (p = .001),multinucleated giant cells (p = .028),tumor cells (p < .001),foamy histiocytes (p = .002)and pigmented histiocytes (p < .001)were associated with a positive histology.Presence of lymphoid fragments (p < .001)and a composition rich in lymphocytes correlated with a negative histologic follow-up (p = .005).Only the presence of tumor cells (p < .001)and lymphoid fragments (p < .001)were independently associated with nodal status Demographics of the cohorts.Comparison of cytologic features between aspirates with and without histologic evidence of metastatic papillary thyroid carcinoma.
T A B L E 2For cytologically negative aspirates, on univariable analysis, presence of colloid material ( p = .010),necrotic material (p = .021),and pigmented histiocytes (p = .024)were associated with a positive node, whereas the presence of lymphoid fragments (p = .002)and a composition rich in lymphocytes ( p = .002)were associated with a negative histology.Multivariable analysis demonstrated the presence of lymphoid fragments (p = .001),colloid material ( p < .001),and pigmented histiocytes ( p = .003)to be significant independent factors, T A B L E 3 T A B L E 4 Comparison of cytologic features in aspirates without tumor cells.