To the Editor, Ectopic intrathyroidal thymic tissue (EITT) occurs due to aberrant cervical migration during embryogenesis. It is a benign and self-limiting condition that is not uncommon in pediatric age group and may present as a thyroid nodule. The immature lymphocytes present in thymoma and T-lymphoblastic lymphoma (T-LBL) may have morphological resemblance to and share some immunophenotypic characteristics with normal thymocytes. Therefore, diagnosis of EITT based on flow cytometry could be challenging.

Normal thymic tissue has a specific T cell maturation pattern; the characteristic CD4+CD8+ double positive (DP) either CD3− or more mature CD3+ thymocytes predominate. On a CD3/CD4/CD8 dot plot, CD3+ thymocytes produce characteristic “classic wing pattern” with CD3+ DP thymocytes forming the body and maturing helper and cytotoxic T-cells, the wings (Li, Dim, Paulson, & Rivard, 2019; Yuan et al., 2018). This typical pattern is useful in differentiating normal thymic tissue from benign thymoma or T-LBL. Here, we describe two cases of EITT in children, one with tuberous sclerosis and the other with Graves' disease, which displayed a unique flow cytometric profile with the presence of tightly clustered immature T cells, raising the concern about a possible T-LBL along with skewed distribution of more mature thymic subsets.

We reviewed clinical features, ultrasonography, fine needle aspiration (FNA) biopsy specimens for histopathology, 10-color flow cytometry and T cell receptor gene (TCR) rearrangement, which was available in one patient. First patient is an 8-year-old euthyroid male with tuberous sclerosis and on melatonin and diphenhydramine (Case #1). Second patient is a 4-year-old male with Graves' disease (Case #2) who is being treated with methimazole and atenolol.

FNA biopsies were carried out after finding a hyperechoic thyroid nodule in Case#1 and soft tissue mass with solid heterogeneous, speckled pattern in Case#2 on ultrasonography. The flow cytometry of FNA biopsies displayed CD45bright lymphocytes consisting of 77% CD3+ mature T cells with 52% CD4+, 21% CD8+ cells and 1% DP with a CD4/CD8 ratio of 2.5. CD3 expression on mature T cell was relatively homogeneous (Figure 1a). Interestingly, it also displayed a tightly clustered population of CD45dim immature T lymphocytes (22%) that were predominantly CD3−, CD5dim+, CD2dim+, CD4partial+, CD8−, CD1a+, and CD34partial+. Fifty-eight percent of gated CD45dim population was CD4+, 19% was CD4+CD8+ (DP) and 42% was CD4−CD8−CD34+ (Figure 1b). Intracytoplasmic CD3 (iCD3) and TdT was not assessed due to insufficient specimen.

Case #2 showed a population of CD45bright lymphocytes consisting of 58% CD3+ mature T cells with 33% CD4+, 18% CD8+ and 1% DP with a CD4/CD8 ratio of 1.8. CD3 expression on mature T cell was relatively homogeneous (Figure 1c). Case #2 also showed a tightly clustered population of CD45dim immature T lymphocytes (32%) that were predominantly iCD3+, surface CD3−, CD5dim+, CD2dim+, CD4partial+, CD8−, CD1a+, CD34partial+ and TdT+. Fifty four percent of gated CD45dim population was CD4+, 5% DP and 46% CD4−CD8−CD34+ (Figure 1d).

Both specimens displayed an absence of CD10 expression on CD34+ immature T cells, decreased presence of DP T cells within CD45dim population and absence of DP maturing T cells. There was no aberrant B cell or myeloid marker expression seen in either case and TCR rearrangement study in Case #2 did not show clonality.

Histopathology of FNA biopsies showed polymorphic lymphocytes admixed with cohesive epithelia groups and lack of thyroid tissue in Case #1, and polymorphic lymphocytes admixed with cohesive epithelial cells with no evidence of papillary thyroid carcinoma in Case #2. Immunohistochemical stains were not feasible in both cases because biopsy specimens were hemodiluted and showed scant number of cells (Figure 1e–h). Both cases were concluded as EITT.

Thymocyte maturation process is greatly regulated by thymic epithelial cells and follows a specific maturation pattern (Yuan et al., 2018). During normal development, early maturing thymocytes are CD3−CD7+CD2+CD5dim+CD4−CD8−CD1a−CD34+. Maturing thymocytes gradually start expressing CD1a, CD4 followed by CD8; CD10 expression is upregulated with simultaneous decrease in CD34 expression. Then, gain of expression of CD3 follows and finally loss of CD10, CD1a and either expression of CD8 or CD4 occurs leading to development of mature helper or cytotoxic T cells. Therefore, normal thymus typically displays CD3−CD34+CD4−CD8− (DN), CD3−CD34−CD4dim+CD8−, CD3−CD34− DP, and CD3+ DP, CD3+CD4+CD8− helper, CD3+CD4−CD8+ cytotoxic T cells subsets with CD3+ thymocytes producing characteristic “classic wing pattern” by flow cytometry. Understanding the characteristic pattern of thymocyte subsets is very important for accurate diagnosis of ectopic thymic tissue.

Primary thyroid T cell lymphomas are extremely rare and can involve thyroid gland without distant metastases. Thymomas are epithelial tumors that contain a large number of immature T cells and are frequently associated with various autoimmune disorder, including Graves' disease. Ectopic thymomas are rare tumors that originate in areas including the thyroid, posterior or middle mediastinum. The flowcytometric features of normal thymus, T-LBL, thymoma and two cases of EITT are described in Table 1.

Two cases of thyroid nodule that we describe showed tight clustering of CD45dim T cells on CD45/side scatter graph and lack the characteristic smearing pattern of CD3. CD45dimCD3− cells were predominantly either single CD4+ or DN. Moreover, there was an absence of CD3−CD34+CD10+ subset and CD3+ DP subset seen in a normal thymus. Although atypical presentation seen in these two cases share some features of T-LBL, first immature cells comprised a fraction of the lymphoid cells in the mixture lacking homogeneity in cellular composition. Second, there were no aberrant B cell or myeloid antigen expression, and no clonal TCR rearrangement at least in one case.

The percentage of DP thymocytes are always greater than 50% in normal pediatric thymus, 40%–95% in types A/B and B thymomas and <20% in type A thymoma (Li et al., 2019). However, type A thymomas are predominantly composed of thymic epithelial cells with only scant immature T lymphocytes. In thymoma, the distribution of maturing thymocytes may be skewed by the absence of normal epithelial support (Yuan et al., 2018). The two cases that we evaluated showed mature/maturing lymphocytes with significantly lower population of CD3− DP cells, absent CD3+ DP subset in more mature cell population, as well as lack of CD34+CD10+ and CD34−CD10+ subsets, differing from an ectopic thymoma profile.

Flow cytometric findings of these two cases presented are unique and appear almost a copy of one and other (Figure 1a–d). The difference in the thymocyte maturation pattern including lack of CD34+CD10+ and CD34−CD10+, significantly decreased CD3− DP cells and absent CD3+ DP subset could be partially due to influence of the host tissue along with absent and/or hypo-/dysfunctional thymic epithelium. In fact, the ectopic thymus tissue flow cytometric profile in our cases is quite different than the reported case in adenoid tissue (Yuan et al., 2018) and also differ from the previously reported EITT cases (Li et al., 2019). Interestingly, both immature CD3− negative and more mature CD3− positive populations were predominated by CD4− positive thymocytes. Peripheral blood contamination as evidenced by granulocyte population on CD45/SS plots may be a factor in increased CD4− positive T cells in mature gate; however, CD4 predominance in immature CD3− negative thymocytes is unusual. Therefore, potential effect of ectopic microenvironment on the final composition of the EITT tissue warrants further investigation.

对编辑来说，异位甲状腺内胸腺组织 (EITT) 是由于胚胎发育过程中异常的宫颈迁移而发生的。这是一种良性和自限性疾病，在儿童年龄组中并不少见，可能表现为甲状腺结节。胸腺瘤和 T 淋巴细胞淋巴瘤 (T-LBL) 中存在的未成熟淋巴细胞可能与正常胸腺细胞具有形态相似性并具有一些免疫表型特征。因此，基于流式细胞术的 EITT 诊断可能具有挑战性。

正常胸腺组织具有特定的 T 细胞成熟模式；特征性 CD4+CD8+ 双阳性 (DP) CD3- 或更成熟的 CD3+ 胸腺细胞占优势。在 CD3/CD4/CD8 点图上，CD3+ 胸腺细胞产生特征性的“经典翼型”，CD3+ DP 胸腺细胞形成机体并成熟辅助性 T 细胞和细胞毒性 T 细胞，翼（Li, Dim, Paulson, & Rivard,  2019 ; Yuan等人，  2018）。这种典型模式可用于区分正常胸腺组织与良性胸腺瘤或 T-LBL。在这里，我们描述了两例儿童 EITT，一例患有结节性硬化症，另一例患有 Graves 病，其表现出独特的流式细胞仪特征，存在紧密聚集的未成熟 T 细胞，引发了对可能存在 T-LBL 的担忧。更成熟的胸腺亚群分布偏斜。

我们回顾了临床特征、超声检查、用于组织病理学的细针抽吸 (FNA) 活检标本、10 色流式细胞术和 T 细胞受体基因 ( TCR ) 重排，这些标本可用于一名患者。第一名患者是一名 8 岁甲状腺功能正常的男性，患有结节性硬化症，服用褪黑激素和苯海拉明（病例 #1）。第二名患者是一名 4 岁男性，患有 Graves 病（病例 #2），正在接受甲巯咪唑和阿替洛尔治疗。

FNA 活检在病例#1 中发现高回声甲状腺结节，在病例#2 中发现软组织肿块，在超声检查中发现实性不均匀、斑点状图案。FNA 活检的流式细胞术显示 CD45bright 淋巴细胞由 77% CD3+ 成熟 T 细胞和 52% CD4+、21% CD8+ 细胞和 1% DP 组成，CD4/CD8 比率为 2.5。成熟 T 细胞上的 CD3 表达相对均匀（图 1a）。有趣的是，它还显示了紧密聚集的 CD45dim 未成熟 T 淋巴细胞群 (22%)，主要是 CD3-、CD5dim+、CD2dim+、CD4partial+、CD8-、CD1a+ 和 CD34partial+。58% 的门控 CD45dim 群体是 CD4+，19% 是 CD4+CD8+ (DP)，42% 是 CD4-CD8-CD34+（图 1b）。由于标本不足，未评估胞浆内 CD3 (iCD3) 和 TdT。

案例 #2 显示了由 58% CD3+ 成熟 T 细胞和 33% CD4+、18% CD8+ 和 1% DP 组成的 CD45bright 淋巴细胞群，CD4/CD8 比率为 1.8。成熟 T 细胞上的 CD3 表达相对均匀（图 1c）。病例 #2 还显示了紧密聚集的 CD45dim 未成熟 T 淋巴细胞群 (32%)，主要是 iCD3+、表面 CD3-、CD5dim+、CD2dim+、CD4partial+、CD8-、CD1a+、CD34partial+ 和 TdT+。54% 的门控 CD45dim 群体是 CD4+、5% DP 和 46% CD4-CD8-CD34+（图 1d）。

两种标本均显示 CD34+ 未成熟 T 细胞上不存在 CD10 表达，CD45dim 群体中 DP T 细胞的存在减少以及 DP 成熟 T 细胞不存在。在任一病例中均未见异常的 B 细胞或骨髓标记表达，并且病例 #2 中的TCR重排研究未显示克隆性。

FNA活检的组织病理学显示，在病例#1中，多形性淋巴细胞与粘性上皮细胞群混合，缺乏甲状腺组织，在病例#2中，多形性淋巴细胞与粘性上皮细胞混合，没有甲状腺乳头状癌的证据。在这两种情况下，免疫组织化学染色都不可行，因为活检标本被血液稀释并且细胞数量很少（图 1e-h）。两起案件均以 EITT 结案。

胸腺细胞成熟过程受到胸腺上皮细胞的极大调节，并遵循特定的成熟模式（Yuan et al.,  2018）。在正常发育过程中，早期成熟的胸腺细胞是 CD3-CD7+CD2+CD5dim+CD4-CD8-CD1a-CD34+。成熟的胸腺细胞逐渐开始表达 CD1a、CD4，然后是 CD8；CD10 表达上调，同时 CD34 表达降低。然后，CD3 的表达增加，最后 CD10、CD1a 和 CD8 或 CD4 的表达缺失，导致成熟的辅助性或细胞毒性 T 细胞的发育。因此，正常胸腺通常显示 CD3−CD34+CD4−CD8− (DN)、CD3−CD34−CD4dim+CD8−、CD3−CD34− DP 和 CD3+ DP、CD3+CD4+CD8− 辅助、CD3+CD4−CD8+具有 CD3+ 胸腺细胞的细胞毒性 T 细胞亚群通过流式细胞术产生特征性的“经典翼型”。了解胸腺细胞亚群的特征模式对于异位胸腺组织的准确诊断非常重要。

原发性甲状腺 T 细胞淋巴瘤极为罕见，可累及甲状腺而无远处转移。胸腺瘤是含有大量未成熟 T 细胞的上皮肿瘤，通常与各种自身免疫性疾病相关，包括格雷夫斯病。异位胸腺瘤是一种罕见的肿瘤，起源于甲状腺、后纵隔或中纵隔等区域。正常胸腺、T-LBL、胸腺瘤和 2 例 EITT 的流式细胞仪特征见表 1。

我们描述的两例甲状腺结节在 CD45/侧向散点图上显示 CD45dim T 细胞紧密聚集，缺乏 CD3 的特征性涂片模式。CD45dimCD3- 细胞主要是单个 CD4+ 或 DN。此外，在正常胸腺中不存在 CD3-CD34+CD10+ 亚群和 CD3+ DP 亚群。尽管在这两种情况下看到的非典型表现具有 T-LBL 的一些特征，但首先未成熟细胞包含混合物中的一部分淋巴细胞，在细胞组成方面缺乏同质性。其次，至少在一个病例中没有异常的B细胞或骨髓抗原表达，也没有克隆TCR重排。

DP 胸腺细胞的百分比在正常儿童胸腺中总是大于 50%，在 A/B 型和 B 型胸腺瘤中为 40%–95%，在 A 型胸腺瘤中 <20% (Li et al.,  2019 )。然而，A型胸腺瘤主要由胸腺上皮细胞组成，只有少量未成熟的T淋巴细胞。在胸腺瘤中，成熟胸腺细胞的分布可能因缺乏正常的上皮支持而出现偏差（Yuan et al.,  2018）。我们评估的两个病例显示成熟/成熟淋巴细胞具有显着较低的 CD3-DP 细胞群，在更成熟的细胞群中不存在 CD3+ DP 亚群，以及缺乏 CD34+CD10+ 和 CD34-CD10+ 亚群，与异位胸腺瘤不同轮廓。

这两个病例的流式细胞术发现是独一无二的，几乎是一个和另一个的副本（图 1a-d）。胸腺细胞成熟模式的差异包括缺乏 CD34+CD10+ 和 CD34-CD10+、显着减少的 CD3-DP 细胞和缺乏 CD3+DP 亚群可能部分是由于宿主组织的影响以及缺乏和/或功能低下/功能失调的胸腺上皮。事实上，我们病例中的异位胸腺组织流式细胞仪谱与腺样体组织中报道的病例有很大不同（Yuan et al.,  2018），也不同于之前报道的 EITT 病例（Li et al.,  2019））。有趣的是，未成熟的 CD3- 阴性和更成熟的 CD3- 阳性群体均以 CD4- 阳性胸腺细胞为主。CD45/SS 图上的粒细胞群证明外周血污染可能是成熟门中 CD4- 阳性 T 细胞增加的一个因素；然而，在未成熟的 CD3- 阴性胸腺细胞中 CD4 占优势是不寻常的。因此，异位微环境对 EITT 组织最终组成的潜在影响值得进一步研究。