| Abstract|| |
Background: Intrathyroid lymphoid tissue is accrued in Hashimoto thyroiditis (HT). Histologically, this acquired lymphoid tissue bears a close resemblance to mucosa-associated lymphoid tissue (MALT) and can evolve to lymphoma. Aim: To demonstrate the morphological, and immunohistochemical profiles of Hashimoto thyroiditis and to ascertain the importance of light chain restriction in distinguishing HT with extensive lymphoplasmacytoid infiltrate from MALT lymphoma. Materials and Methods: We studied histopathologically and immunohistochemically (CD20, CD3, Igk, Igl and cytokeratin) 30 cases of HT for evaluation of the lymphoid infiltrate and the presence of lymphoepithelial lesions (LELs). Distinguishing between early thyroid lymphoma and HT was evaluated by light chain restriction. These findings were compared with two cases of primary thyroid lymphoma. Results: The histopathological findings were characteristic of HT. Immunohistochemistry confirmed inconspicuous, rare B-cell LELs as well as a prominent T-lymphocyte population. Testing for light chain restriction showed polyclonal population of plasma cells. The cases of MALT lymphoma had distinct destructive lymphoepithelial lesions, B-cell immunophenotyping and showed kappa light chain restriction in the plasmacytoid population. Conclusions: Hashimoto thyroiditis differs both histopathologically and immunohistochemically from thyroid lymphoma. In suspicious cases, immunohistochemistry could be helpful in reaching a definitive diagnosis.
Keywords: Hashimoto thyroiditis, immunohistochemistry, primary thyroid lymphoma
|How to cite this article:|
Amani H K. Histopathologic and immunohistochemical features of Hashimoto thyroiditis. Indian J Pathol Microbiol 2011;54:464-71
Several studies have linked certain autoimmune and chronic inflammatory conditions to an increased occurrence of lymphoma.  However, the magnitude of the average lymphoma risk increase in each disorder varies considerably among studies.  It is well known that almost all thyroid lymphomas arise in the setting of Hashimoto thyroiditis (HT), which induces reactive lymphoid proliferation leading to the development of mucosa-associated lymphoid tissue (MALT) lymphoma, which can lead to an aggressive lymphoma. ,,,,,,
We need to learn how to identify individuals who are at substantially increased lymphoma risk and how to intervene against this risk. 
The coexistence of reactive and neoplastic processes in the thyroid may cause difficulty in diagnosing mucosa associated lymphoid tissue lymphoma (MALTOMA) using cytology or histology. This has led to the use of immunohistochemistry and molecular techniques to confirm or exclude the diagnosis. 
The aims of our study were to demonstrate the morphological and immunohistochemical profiles of HT, and to ascertain the importance of clonality in distinguishing HT from MALT lymphoma.
We used morphology and immunohistochemistry to study 30 cases of HT. Clonality was assessed by immunostaining for kappa (k) and lambda (l) light chains of immunoglobulins (Ig). The findings were compared with two cases of primary thyroid lymphoma.
| Materials and Methods|| |
The present study was performed on 32 thyroidectomy specimens corresponding to 30 cases of HT and two cases of primary thyroid non-Hodgkin's lymphoma, received at the Pathology Department of the Medical Research Institute, Alexandria University, Egypt. The procedures followed in the study were in accordance with the ethical standards of the Medical Research Institute committee on human experimentation.
Formalin-fixed, paraffin-embedded 3 to 4-mm tissue sections were prepared from each case and stained routinely by hematoxylin and eosin (H and E) stain for histopathologic diagnosis and evaluation of the lymphoid infiltrate, and the presence of lymphoepithelial lesions (LELs). Lymphoepithelial lesions were defined as clusters of three or more lymphocytes in the glandular epithelium.
Further sections on coated slides were prepared for immunohistochemical staining using avidin biotin peroxidase complex method.  Briefly, after the inhibition of endogenous peroxidases with 3% hydrogen peroxide, slides were treated by microwave in 10mM citrate buffer for 15 min. After washing the slides in Tris-Buffer saline (TBS), they were incubated with 10% normal goat serum at 23°C for 30 min. Slides were then incubated at 4°C overnight with the following primary antibodies: CD20 (L-26), CD3, Igk, Igl and anti-cytokeratin antibody (AE1/AE3) (Dako, Denmark).
The incubation was followed by addition of biotin-conjugated goat anti-mouse, anti-rabbit IgG diluted 1:200 for 20 min. A final 45-min incubation with streptavidin-conjugated horseradish peroxidase diluted 1:200 in TBS was performed. The reaction product was developed for 5 min with diaminobenzidine tetrahydrochloride (DAB) and the sections were lightly counterstained with Mayer's Hematoxylin.
Monoclonality is assumed when there is a ratio of 10:1 or greater between [kappa] and [lambda] staining cells. 
| Results|| |
A summary of the clinicopathologic characteristics is shown in [Table 1].
|Table 1: Clinicopathologic features and clonality results of 30 patients with Hashimoto's thyroiditis and two patients with primary thyroid lymphoma|
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The present study was undertaken on 30 cases of HT. Twenty-five patients were females. The mean age at diagnosis was 44.63 years (range, 15-70 years). All patients presented with goiter (18 cases diffuse and 12 nodular) of average size 3.7 cm. Two female patients aged 65 and 80 years with primary thyroid lymphoma were included as a control, they presented with a rapidly growing fixed nodular goiter (6 x 5 x 4 cm) and (8 x 5 x 3 cm), extending beyond the thyroid with clinical consequences such as pressure symptoms in the neck and dyspnea.
All cases showed classic features of HT; a lymphoid infiltrate arranged in lymphoid follicles with interfollicular small round lymphocytes, plasma cells, scattered lymphoplasmacytoid cells, and a few large transformed cells [Figure 1]a and b. Most lymphoid follicles had a well-defined germinal centre and mantle zone with absent marginal zone. An extensive lymphoplasmacytoid infiltrate with focal effacement of architecture by atypical cells was seen in two cases with atrophy of thyroid follicles [Figure 1]c. Hurthle cell metaplasia was a constant feature [Figure 1]d. Lymphoepithelial-like lesions were rarely seen [Figure 1]e.
|Figure 1: H and E stain: (a) Hashimoto thyroiditis showing reactive germinal centre beside atrophic thyroid follicles with interfollicular lymphoplasmacytic inflammatory infi ltrate (x400). (b) Hashimoto thyroidi&tis showing lymphoplasmacytic infiltrate with focal LELs (x400). (c) Effacement of thyroid architecture in Hashimoto thyroiditis by the lymphoplasmacytic infiltrate. Note residual thyroid follicles (arrows) x400). (d) Hurthle cell metaplasia of follicular epithelium in Hashimoto thyroiditis (x400). (e) Occasional LELs (arrows) in Hashimoto thyroiditis (x400)|
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Primary thyroid lymphoma
In the cases of primary thyroid lymphoma, the parenchyma was diffusely replaced by a dense, atypical lymphoproliferative infiltrate, composed of monocytoid lymphocytes with small, slightly irregular folded or centrocyte-like nuclear contours, condensed nuclear chromatin, inconspicuous nucleoli, and abundant pale cytoplasm [Figure 2]a. The lymphoepithelial lesions were frequent [Figure 2]b. An admixture of lymphoplasmacytoid cells having abundant, eccentrically placed brightly eosinophilic cytoplasm was identified. Residual thyroid epithelium was present in the lymphoid infiltrate; this took the form of small follicles containing colloid or of small acinar structures without colloid. The capsule was invaded as well as the adjacent adipose tissue and skeletal muscles.
|Figure 2: Cases of MALT thyroid lymphoma: (a) Monocytoid and plasmacytoid lymphocytes (arrows). (H and E stain X400). (b) LELs (H and E stain x400). (c) CD20-positive tumor cells (Immunoperoxidase stain; x400). (d) CD3-negative tumor cells (Immunoperoxidase stain; x400). (e) Residual thyroid follicles (arrow). (CD20 immunohistochemistry; x400). (f) Destructive LELs by CD20-positive tumor cells with MALT ball (arrow) (Immunoperoxidase stain; x400). (g) Few residual thyroid follicles stained by anti cytokeratin; note the LEL (arrow) (Immunoperoxidase stain; x400)|
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Hashimoto thyroiditis : Was composed of numerous small lymphocytes (a mixture of T and B-cells, but often with a predominance of T-cells). Most of the lymphoid infiltrate in HT was arranged as lymphoid follicles composed of B-cells (CD20+, CD3-) [Figure 3]a. The interfollicular lymphoid infiltrate was composed predominantly of T-cells (CD3+, CD20-) admixed with a very small number of B-cells and plasma cells [Figure 3]b-e. In the scant lymphoepithelial lesions seen, the invading lymphocytes were T-cells (CD3+) [Figure 3]f. Cytokeratin demonstrated the preserved thyroid follicles [Figure 3]g. The two lymphocyte rich thyroiditis cases showed increase in CD20-positive lymphocytes with well-circumscribed germinal centers surrounded by mantle zone. The follicles were connected by interfollicular dense lymphoid infiltrate widely separating the follicular acini [Figure 3]h. However, the lymphoid population was polyclonal as evidenced by dual positivity for k and l immunostaining. In these polyclonal infiltrates, the lambda/kappa ratio never exceeded 1:5. [Figure 4]a and b.
|Figure 3: Immunoperoxidase stain in HT: (a) Reactive lymphoid follicles decorated by CD20 (x200). (b) Predominance of CD3-positive T-cells (x200). (c) HT with reactive lymphoid follicles having CD3-posi&tive mantle zone (x200). (d) Few CD20-positive B-cells (x400). (e) CD3-positive T-cell infi ltrate in between the thyroid follicles (x400). (f) Few non-destructive LELs (arrow) formed of CD3-positive T-cells (x400). (g) Intact thyroid follicles decorated by cytokeratin (x200). (h) Frequent CD20-positive lymphoid follicles and interstitial lymphoplasmacytoid infi ltrate (x200)|
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|Figure 4: Immunoperoxidase stain (a) Heterogeneous positivity for K immunostaining (arrow) in Hashimoto thyroiditis (x400). (b) Hashimoto thyroiditis showing positive λ immunostaining of the reactive lymphoid population (arrow), the arrowhead points to thyroid follicle with small non-destructive LEL (x400). (c) Thyroid lymphoma; all tumor cells show strong and diff use expression of K light chain (x400). (d) Thyroid lymphoma; all tumor cells show lack of λ light chain expression (x400)|
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Primary thyroid lymphoma: The two cases of MALT lymphoma were characterized by predominance of B-cells that were not confined to germinal centers. The small and large neoplastic cells in the marginal zone and in the diffuse areas expressed CD20, and were negative for CD3 confirming B-cell lineage [Figure 2]c and d. Rare intact thyroid follicles were identified due to the abundance of destructive lymphoepithelial lesions [Figure 2]e. These lymphoepithelial lesions were more numerous than in HT cases and showed prominent follicular stuffing, with focal packing of follicular lumina by centrocyte-like lymphoid cells [Figure 2]f. Cytokeratin decorated the residual atrophic thyroid follicular elements [Figure 2]g and h. Immunostaining for k and l confirmed the morphologic diagnosis, where the lymphoid population was clear-cut monoclonal for k with at least a 10:1 ratio, counted in 10 high-power fields [Figure 4]c and d.
| Discussion|| |
Hashimoto thyroiditis is an established risk factor for the development of lymphoma in the thyroid.  These patients have a threefold excess risk of developing lymphoma, and an 80 fold increased risk of thyroid lymphoma (the overall lymphoma risk is 0.1% of patients).  Indeed, lymphocytic thyroiditis is present in the background in 94% of thyroid lymphomas.  Even if endocrinologists are aware of the increased risk of lymphoma in these pathologies, diagnosis of thyroid lymphoma remains difficult because the clinical history, the physical examination, the thyroid function tests, and the ultrasound examination are not specific to detect the occurrence of lymphoproliferative disorders. Furthermore, it is sometimes difficult to recognize the low-grade lesions as distinct from lymphocytic thyroiditis. Distinguishing severe chronic lymphocytic thyroiditis from lymphoma can, at times, pose great difficulty. 
The present work was undertaken on 30 cases of HT in order to characterize them, both morphologically and by immunohistochemistry. Two cases of MALT thyroid lymphoma were studied in parallel by the same methods, in order to highlight and establish the difference between them. The aims of the work were to find the precise criteria that could enable us to differentiate between HT and early lymphoma. The second aim was to determine if monoclonal lymphoid population could occur in otherwise bland HT, as it can occur in Helicobacter pylori-associated gastritis and Sjφgren disease. ,
In this study, cases of HT were characterized by frequent lymphoid follicles with reactive wide germinal centers. The mantle zone was prominent and the interfollicular tissue was widened by a dense infiltrate of lymphocytes and plasma cells. The thyroid follicles showed atrophy and Hurthle cell metaplasia. Two cases showed partial effacement of the thyroid architecture by the lymphoid infiltrate with focal lymphoepithelial-like lesions; however, areas typical of HT often predominate. Similar findings have been reported in the literature,  where it was noted that thyroiditis can create an effacement of the normal thyroid follicular architecture, even with the formation of what could be classified as lymphoepithelial lesions. Several researches , indicated that the germinal centers, often a prominent component of thyroiditis, can be scanty and appear over-run in some severe cases.
A predominant reactive lymphoid population might mask an early lymphomatous transformation. On the other hand serologic markers such as lactic dehydrogenase (LDH) and ί 2 -microglobulin became significantly elevated only in advanced lymphomas. Therefore, a thyroid biopsy would be more appropriate in such a situation to reach the proper diagnosis. ,
In accord with others, , all the cases of HT studied in this work showed an admixture of B and T-lymphocytes with the latter predominating. CD20 highlighted the germinal centers, while CD3 demonstrated the well-developed mantle zone as well as the interfollicular population. The cases showing apparent lymphoepithelial lesions were formed mainly of T-lymphocytes thus excluding MALT lymphoma. Cytokeratin was also done which indicated the rarity of destructive lymphoepithelial lesions in thyroiditis cases.
In the literature others have detected clonal B-cell proliferation in HT by several means (immunohistochemistry (IHC), southern blot hybridization, polymerase chain reaction (PCR), and PCR + sequencing). ,,,, Some explained the presence of such clones as a selective proliferation of a small number of B-cell clones as part of the autoimmune response in HT. , Others confirmed that thyroid lymphomas do develop in patients previously diagnosed as HT.  Furthermore, D'Antonio et al., have reported the existence of a minute focus of extranodal marginal zone lymphoma about 4 mm in diameter in a case of HT, and recommended careful examination of thyroid specimens to disclose small foci of lymphomatous transformation.
In the present study two cases of HT showed focal effacement of architecture by atypical lymphocytes. Therefore, the possibility is raised that these cases might be harboring a clone that may later on develop into lymphoma. However, k and l immunostaining demonstrated polyclonal staining of lymphocytes, meaning that the infiltrating lymphocytes were a reactive proliferation, thus excluding the possibility of early lymphoma.
In accord with others, the two cases of MALT lymphoma included in this work were characterized by effacement of thyroid architecture by diffuse lymphoid infiltrate extending to extrathyroid tissues and the infiltrate mainly composed of monocytoid cells having cleaved nuclei and abundant clear cytoplasm. Admixed were plasmacytoid lymphocytes. , Destructive LELs were abundant, completely replacing the thyroid follicles. The residual follicles were highlighted by anticytokeratin, CD20 was uniformly positive, whereas CD3 was negative, confirming the B-cell nature of lymphoma. Kappa and lambda immunostaining revealed strong homogenous positivity for k immunostaining and negative l immunostaining.
In conclusion, strict morphological and immunohistochemical criteria differentiate HT from MALT lymphoma. For morphologically borderline cases (florid lymphoid infiltrate), cytokeratin, CD20, CD3 in addition to k and l immunostaining could contribute in making the distinction.
| References|| |
|1.||Leandro MJ, Isenberg DA. Rheumatic diseases and malignancy-is there an association? Scand J Rheumatol 2001;30:185-8. |
|2.||Zintzaras E, Voulgarelis M, Moutsopoulos HM. The risk of lymphoma development in autoimmune diseases: A meta-analysis. Arch Intern Med 2005;165:2337-44. |
|3.||Cabay RJ, Salem F. Chronic lymphocytic (Hashimoto) thyroiditis: An interesting cellular aggregate in fine-needle aspiration biopsy. Diagn Cytopathol 2009;37:191. |
|4.||Holm L, Blomgren H, Lowhagen T. Cancer risks in patients with chronic lymphocytic thyroiditis. N Engl J Med 1985;312:601-4. |
|5.||Hyjek E, Isaacson P. Primary B-cell lymphoma of the thyroid and its relationship to Hashimoto's thyroiditis. Hum Pathol 1988;19:1315-26. |
|6.||Isaacson PG, Androulakis-Papachristou A, Diss TC, Pan L, Wright DH. Follicular colonization in thyroid lymphoma. Am J Pathol 1992;141:43- 52. |
|7.||Isaacson P. The MALT lymphoma concept updated. Ann Oncol 1995;6:319-20. |
|8.||Peng H, Du M, Diss T, Isaacson P, Pan L. Genetic evidence for a clonal link between low and high-grade components in gastric MALT B-cell lymphoma. Histopathology 1997;30:425-9. |
|9.||Chan J, Ng C, Isaacson P. Relationship between high-grade lymphoma and low-grade B-cell mucosa-associated lymphoid tissue lymphoma (MALToma) of the stomach. Am J Pathol 1990;136:1153-64. |
|10.||Smedby KE, Baecklund E, Askling J. Malignant Lymphomas in Autoimmunity and Inflammation: A Review of Risks, Risk Factors, and Lymphoma Characteristics. Cancer Epidemiology Biomarkers and Prevention 2006;15:2069-77. |
|11.||Wozniak R, Beckwith L, Ratech H, Surks MI. Maltoma of the Thyroid in a Man with Hashimoto's Thyroiditis. J Clin Endocrinol Metab 1999;84:1206-9. |
|12.||Hsu SM, Rante M, Fauger H. Use of Avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577-80. |
|13.||Burg G, Kerl H, Kaudewitz P, Braun-Falco O, Mason DY. Immunoenzymatic typing of lymphoplasmacytoid skin infiltrates. J Dermatol Surg Oncol 1984;10:284-90. |
|14.||Derringer GA, Thompson LD, Frommelt RA, Bijwaard KE, Heffess CS, Abbondanzo SL. Malignant lymphoma of the thyroid gland: A clinicopathologic study of 108 cases. Am J Surg Pathol 2000;24:623- 39. |
|15.||Thieblemont C, Mayer A, Dumontet C, Barbier Y, Callet-Bauchu E, Felman P, et al. Primary Thyroid Lymphoma Is a Heterogeneous Disease. J Clin Endocrinol Metab 2002;87:1105-11. |
|16.||Hsi ED, Singleton TP, Svoboda SM, Schnitzer B, Ross CW. Characterization of the lymphoid infiltrate in Hashimoto thyroiditis by immunohistochemistry and polymerase chain reaction for immunoglobulin heavy chain gene rearrangement. Am J Clin Pathol 1998;110:327-33. |
|17.||Ben-Ezra J, Wu A, Sheibani K. Hashimoto's thyroiditis lacks detectable clonal immunoglobulin and T cell receptor gene rearrangement. Hum Pathol 1988;19:1444-8. |
|18.||Soma L, Bobzien B, Guerin C, LiVolsi V. Pathologic Quiz Case: A Rapidly Enlarging Thyroid. Arch Pathol Lab Med 2003;127: e253-4. |
|19.||Ohye H, Fukata S, Kubota S, Sasaki I, Takamura Y, Matsuzuka F, et al. Techniques in Thyroidology, Successful Treatment for Recurrent Painful Hashimoto's Thyroiditis by Total Thyroidectomy. Thyroid 2005;15:340- 5. |
|20.||Matsuzuka F, Fukata S, Kuma K, Miyauchi A, Kakudo K, Sugawara M. Gene rearrangement of immunoglobulin as a marker of thyroid lymphoma. World J Surg 1998;22:558-6. |
|21.||Takano T, Miyauchi A, Matsuzuka F, Yoshida H, Kuma K, Amino N. Diagnosis of thyroid malignant lymphoma by reverse transcription-polymerase chain reaction detecting the monoclonality of immunoglobulin heavy chain messenger ribonucleic acid. J Clin Endocrinol Metab 2000;85:671-6. |
|22.||Saxena A, Alport EC, Moshynska O, Kanthan R, Boctor MA. Clonal B cell populations in a minority of patients with Hashimoto's thyroiditis. J Clin Pathol 2004;57:1258-63. |
|23.||Knowles DM, Athan E, Ubriaco A. Extranodal noncutaneous lymphoid hyperplasia represents a continuous spectrum of B-cell neoplasia: Demonstration by molecular genetic analysis. Blood 1989;73:1635-45. |
|24.||Tiemann M, Asbeck R, Wacjer H. Clonal B-cell reaction in Sjogren's disease and Hashimoto's autoimmune thyroiditis. Pathologe 1996;17:289-95. |
|25.||Zeppa P, Cozzolino I, Peluso AL, Troncone G, Lucariello A, Picardi M, et al. Cytologic, Flow cytometry and molecular assessment of lymphoid infiltrate in fine-needle cytology sample of Hashimoto thyroiditis. Cancer Cytopathol 2009;117:174-84. |
|26.||Chen HI, Akpolat I, Mody DR, Lopez-Terrada D, De Leon AP, Luo Y, et al . Restricted k/l light chain ratio by flow cytometry in germinal center B cells in Hashimoto thyroididtis. Am J Clin Pathol 2006;125:42-8. |
|27.||Moshynska OV, Saxena A. Clonal relationship between Hashimoto thyroiditis and thyroid lymphoma. J Clin Pathol 2008;61:438-44. |
|28.||D'Antonio A, Caleo A, Licci S, Addesso M, De Palma M, Boscaino A, et al. Thyroid Res 2009;2:9. |
H Kazem Amani
Deaprtment of Pathology, Medical Research Institute, Alexandria University, 67, Omar Loutfi Street, Alexandria
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]