Identification of Cytological Features Distinguishing Mucosa-Associated Lymphoid Tissue Lymphoma from Reactive Lymphoid Proliferation Using Thyroid Liquid-Based Cytology

Liquid-based cytology (LBC) has recently become increasingly popular for thyroid aspirates [1]. LBC specimens have been used to identify some diagnostic clues for papillary thyroid carcinoma (PTC), nodular goiter, and follicular neoplasm, such as convoluted nuclei, intercellular spaces, cytoplasmic changes, and membranous material [2, 3]. However, cytological findings from conventional and LBC specimens have shown some differences [4-6].

Primary thyroid lymphomas are usually non-Hodgkin B-cell lymphomas and reveal preexisting Hashimoto thyroiditis [7, 8]. There are two types of lesions: the aggressive, diffuse large B-cell cell lymphoma (DLBCL) and the indolent, mucosa-associated lymphoid tissue lymphoma (MALT-L) [9]. Cytological diagnosis of DLBCL is not difficult [10]. However, MALT-L is frequently confused with Hashimoto thyroiditis associated with prominent lymphoid proliferation [11-14]. Thus far, the cytological findings for both have been described only in conventional specimens. To the best of our knowledge, there are no reports describing the cytological findings of primary thyroid lymphomas using LBC specimens. Therefore, in this study, we aimed to clarify the cytological differences between MALT-L and reactive lymphoid proliferation using thyroid LBC specimens.

We reviewed the pathology report database of 70 MALT-L cases, 13 DLBCL cases, 61 Hashimoto thyroiditis cases with prominent lymphoid proliferation, and 148 PTC cases with prominent lymphocytic infiltration. All of the patients were operated on at the Kuma Hospital from January 2013 to November 2016. The diagnosis of MALT-L was based on the following criteria: (1) diffuse proliferation of medium-sized CD20-positive lymphoid cells, (2) packing or lymphoepithelial lesion composed of CD20-positive lymphoid cells and follicular cells, (3) follicular colonization confirmed by structural alterations in the meshwork of CD23-positive follicular dendritic cells, and (4) at least one of restricted light chain (κ/λ ratio > 3 or < 0.33) by flow cytometry, chromosomal abnormality by G-banding, or immunoglobulin heavy chain gene rearrangements [15]. The diagnosis of DLBCL was based on diffuse proliferation of large CD20-positive lymphoid cells. Cases with none of the above findings were diagnosed as Hashimoto thyroiditis with prominent lymphoid proliferation. Data from 35 patients with MALT-L, 3 with DLBCL, 19 with PTC with prominent lymphocytic infiltration, and 25 with Hashimoto thyroiditis with prominent lymphoid proliferation who underwent preoperative fine needle aspiration cytology and had both conventional and LBC specimens prepared were included. Conventional specimens were prepared by expressing the aspirated material from the needle onto slide glasses and compressing on a second slide, followed by immediate fixation using a cytological fixative. Subsequently, the needles were rinsed gently with 6 mL of CytoRich^TM RED collection fluid (Becton, Dickinson, Franklin Lakes, NJ, USA). LBC specimens were prepared by the CytoRich^TM hand method (Becton, Dickinson). Both types of specimens were simultaneously stained using the Papanicolaou method.

We observed conventional and LBC specimens under ×200 magnification. We examined the following cytological aspects: colloid material, multinucleated giant cells, lymphoglandular bodies, oxyphilic cell clusters, and lymphoid cells. The presence of oxyphilic cell clusters was determined when more than 4 clusters composed of oxyphilic cells were observed on 1 slide. Lymphoid cells included both lymphoma cells and nonneoplastic lymphocytes. We defined nuclei with a size > 10 μm in the major axis as large nuclei. To measure the nuclear size, we used WinROOF image processing software for Windows (Mitani, Tokyo, Japan), and at least 100 lymphoid cells were measured in each case. Swollen naked nuclei were defined as enlarged, naked nuclei with less punctate chromatin patterns and thin nuclear margins. We assessed the statistical significance of the data using Fisher’s exact probability test and the independent-sample t test. p < 0.05 was considered statistically significant.

In LBC specimens, colloid material and oxyphilic cell clusters were seen in 22.9 and 42.9% of MALT-L cases, respectively (Table 1). This incidence was lower than that in Hashimoto thyroiditis with prominent lymphoid proliferation. The differences were statistically significant (p < 0.05, p < 0.001). In conventional specimens, the frequency of lymphoglandular bodies was significantly higher in MALT-L (68.6%) than in Hashimoto thyroiditis with prominent lymphoid proliferation (20.0%) (p < 0.001). Oxyphilic cell clusters were seen in 20.0% of MALT-L cases in conventional specimens, and the incidence was significantly lower than that in Hashimoto thyroiditis with prominent lymphoid proliferation (76.0%; p < 0.001). Comparison between LBC and conventional specimens of MALT-L revealed that there were fewer lymphoglandular bodies in LBC specimens than in conventional specimens (p < 0.001). In conventional specimens, most nonoxyphilic follicular cells appeared to have naked nuclei with a dense chromatin pattern and looked like lymphoid cells. In LBC specimens, they had a distinct cytoplasm, conspicuous nucleoli, and granular chromatin pattern, and could be easily identified (Fig. 1).

The mean nuclear size in MALT-L was 7.7 and 5.5 µm in LBC specimens and conventional specimens, respectively (Table 2). The nuclei in LBC specimens appeared significantly larger than those in conventional specimens (p < 0.001). The mean nuclear size of lymphocytes in Hashimoto thyroiditis with prominent lymphoid proliferation and PTC with prominent lymphocytic infiltration in LBC specimens was 5.8 and 5.6 µm, respectively, and the nuclei appeared significantly larger in LBC specimens than in conventional specimens (p < 0.001 and p < 0.01, respectively) and were significantly smaller than those in MALT-L (p < 0.001). In 62.9% of MALT-L and all DLBCL cases, large nuclei were present in > 10% of lymphoid cells in LBC specimens. One (4.0%) case of Hashimoto thyroiditis with prominent lymphoid proliferation and 1 (5.3%) case of PTC with prominent lymphocytic infiltration exhibited these findings. In conventional specimens, large nuclei (present in > 10% of lymphoid cells) were not observed in MALT-L, Hashimoto thyroiditis with prominent lymphoid proliferation, and PTC with prominent lymphocytic infiltration. Swollen naked nuclei (Fig. 1) were seen in 92.1% of lymphoma cases and 20.5% of benign lymphocyte cases in LBC specimens (p < 0.001). In conventional specimens, the findings were not seen in any of the lesions examined. In LBC specimens, elongated nuclei with a ratio of the major axis to the minor axis > 3 (Fig. 2) and cleaved nuclei with a deep cleft in the nuclear margin (Fig. 3) were seen in 85.7 and 68.6% of MALT-L cases, and in all and 66.7% of DLBCL cases, respectively. Five (20.0%) cases of Hashimoto thyroiditis with prominent lymphoid proliferation showed elongated and cleaved nuclei. No cases of PTC with prominent lymphocytic infiltration showed elongated nuclei. One (5.3%) case with PTC with prominent lymphocytic infiltration showed cleaved nuclei.

The most common subtype of primary thyroid lymphoma is DLBCL, followed by MALT-L [16]. According to Sangalli et al. [17], in cytological specimens, DLBCL shows a rich population of monotonous noncohesive, large-sized cells with moderate or abundant cytoplasm and prominent nucleoli. The diagnosis of lymphoma in DLBCL cases is not difficult. However, MALT-L shows a mixture of small- to medium-sized atypical lymphocytes, monocytoid B cells, immunoblasts, and plasma cells. Thus, it is frequently difficult to distinguish MALT-L from chronic thyroiditis [11-14]. All of these findings have only been observed for conventional specimens, and there are no reports describing the cytological findings of primary thyroid lymphoma using LBC specimens.

Ford et al. [18] described that lymphoid cells have a tendency to aggregate in LBC specimens and might be erroneously considered as follicular cells. However, in our study, follicular cells showed granular chromatin patterns and were quite different from lymphoid cells. This discrepancy may be due to the LBC methods used. They used the ThinPrep® method, but we used the CytoRich^TM hand method. Therefore, we believe that LBC specimens are more useful for distinguishing between lymphoid and follicular cells.

Lymphoglandular bodies are cytoplasmic fragments and are a diagnostic clue for MALT-L [13]. In our study, they were more frequently seen in MALT-L than in Hashimoto thyroiditis with prominent lymphoid proliferation in conventional specimens. However, in LBC specimens, they were seen in only 11.4% of MALT-L cases. Therefore, we believe that lymphoglandular bodies are not reliable as indicators for lymphoma diagnosis in LBC specimens.

Oxyphilic cell clusters associated with abundant lymphocytes are clues for the diagnosis of chronic thyroiditis [12] and are not seen in primary thyroid lymphoma [19]. In our study, oxyphilic cell clusters were observed in 92.0% of cases of Hashimoto thyroiditis with prominent lymphoid proliferation in LBC specimens. However, 42.9% of MALT-L specimens exhibited them. Thus, our results indicate that the presence of oxyphilic cell clusters cannot be used to rule out the possibility of MALT-L.

In previous reports, the nuclear and cytoplasmic sizes of follicular cells and well-differentiated thyroid carcinoma cells observed in LBC specimens were smaller than those in conventional specimens because the fixative used for LBC specimens contains formaldehyde [2, 3, 5]. In our study, the nuclear sizes in LBC specimens were larger than those in conventional specimens in both lymphoma cells and nonneoplastic lymphocytes. To the best of our knowledge, such findings have not been reported previously. Morphologically, the enlarged lymphoid cells in LBC specimens were degenerative and swollen. This phenomenon seems to be due to the fragility of the cells against the fixatives used in the LBC method. The findings were more remarkable in lymphoma cells than in nonneoplastic lymphocytes. Furthermore, in 62.9% of MALT-L and all DLBCL cases, large nuclei were present in > 10% of lymphoid cells in LBC specimens. Only 1 case of Hashimoto thyroiditis with prominent lymphoid proliferation and 1 case of PTC with prominent lymphocytic infiltration showed these findings. These results indicate that the large nuclei defined in this study might be an important finding indicating lymphoma in LBC specimens.

Moreover, the large nuclei were associated with degenerative changes. The nuclei were naked and exhibited less punctate chromatin patterns and thin nuclear margins. These swollen naked nuclei were noted in 92.1% of lymphoma cases and in 20.5% of nonlymphoma cases in LBC specimens. In conventional specimens, these findings were not observed in either lymphoma or nonlymphoma cases. Thus far, these findings have not been described in any study. In the CytoRich^TM methodology, the aspirated cells are already fixed before centrifugation and smearing. Therefore, we believe that these nuclear alterations had already occurred at the time of fixation and are due to the effect of the fixative, CytoRich^TM RED collection fluid. Lymphoma cells seem to be more fragile than nonneoplastic lymphocytes when exposed to the fixative. We consider that the swollen naked nuclei are characteristic of LBC specimens and that their extensive appearance could be a novel indicator for a cytological diagnosis of lymphoma in LBC specimens using the CytoRich^TM hand method.

Cleaved nuclei have been observed in lymphoma cells, particularly in B-cell lymphomas [20, 21]. We found that cleaved nuclei appeared significantly more often in lymphoma cells than in nonneoplastic lymphocytes, but the incidence in LBC specimens was lower than that in conventional specimens. We also found that elongated nuclei were significantly more apparent in primary thyroid lymphoma than in nonneoplastic lymphocytes, and that the incidence in LBC specimens was significantly higher than that in conventional specimens. Therefore, we consider that elongated nuclei are more useful than cleaved nuclei in distinguishing between primary thyroid lymphoma and nonneoplastic lymphocytes in LBC specimens.

In conclusion, our findings indicate that large nuclei with a major axis of > 10 μm present in > 10% of lymphoid cells, elongated nuclei, and markedly swollen naked nuclei are important clues for distinguishing thyroid primary lymphoma from nonneoplastic lymphocytes on LBC specimens. Furthermore, we also showed that LBC specimens help to distinguish between lymphoid and follicular cells. Lymphoglandular bodies are not reliable indicators for making decisions on lymphoma diagnosis using LBC specimens. However, if methods other than the CytoRich^TM hand method are used, the findings may be different; this aspect needs to be addressed in the future.

We would like to thank Editage (www.editage.jp) for English language editing.

Our study conforms to the guidelines of the Ethics Committee of the Kuma Hospital, and all subjects gave informed consent.

There are no conflicts of interest to declare.