Patients with chronic lymphocytic leukemia (CLL) are prone to infectious complications, including ear, nose, and throat (ENT) infections, due to humoral immunodepression and/or immunosuppression related to therapy. However, specific CLL infiltration in nonlymphoid regions of the head and neck causing ENT symptoms but unrelated to an infection has not been described. CLL is a disease of lymphoid organs, but recent studies have reported extranodal localization of the disease involving the skin and the lungs.^{1, 2} Here, we report a series of cases with specific localizations of CLL cells into the mucosa of the rhinopharynx manifesting as symptoms of ENT infection. To gain insight into the characteristics of this entity of CLL, we retrospectively analyzed the clinical, histological, and molecular features of 25 patients with specific ENT involvement.

All patients were followed in the Hematology Department for proven CLL, and they began to present ENT manifestations at a median time after CLL diagnosis of 2.5 years [1–11 years]. At time of the first ENT symptoms, all patients presented and elevated absolute lymphocyte count (mean at 33 × 109/L [9–117]) and an involvement of the lymphoid areas with enlarged lymph nodes and/or splenomegaly in virtually all cases (21 patients, 83%). ENT symptoms included chronic coughing (44%), antero-posterior nasal discharge (44%), nasal congestion (33%) and pharyngitis (33%). These symptoms were related to upper airway infections, but the manifestations were recurrent and persisted after antibiotic therapy. Thorough ENT examination was carried out and revealed an unusual granular aspect of the mucosa in more than half of the cases (56%), showing pavimentous lesions, thickened, swollen, salmon-colored, and localized mostly at the posterior pharyngeal wall.

Given the persistence of ENT symptoms despite antibiotic treatment and without signs of infection, all patients underwent biopsy of the nasal or pharyngeal mucosa (oropharynx in 17 cases, nasopharynx in five cases, tonsils in three cases, and sinuses in two cases). Histology and immunochemistry analysis showed infiltration of small lymphocytes with CLL phenotype expressing the CD20, the CD5 and the CD23 markers. The pattern of infiltration was diffuse in 63% of the cases and perivascular in 37% (Figure 1A), consistent with specific involvement of CLL cells in nasal-associated lymphoid tissue (NALT-CLL).

Regarding the clinical outcome of the patients with NALT-CLL, most of them had poor prognostic features. The majority of the cases were IGHV unmutated (n = 18/25, 72%). No bias of the IGHV repertoire and no CDR3 stereotype was detected. Furthermore, all cases progressed according to International Workshop on CLL criteria (i.e., development of, or worsening of, anemia and/or thrombocytopenia, progressive or symptomatic splenomegaly and/or lymphadenopathy, lymphocyte doubling time <6 months).³ For 13 patients, the manifestations were diagnosed before frontline treatment and they needed to be treated within 2 years after the first ENT symptoms. For the 12 remaining patients, the ENT symptoms occurred at relapse. This indicates that NALT-CLL is associated with disease that is more progressive. Interestingly, the systemic treatment led to a complete regression of the nasopharyngeal symptoms in all patients, further confirming the clonal relation between the ENT lesions and the hematological disease.

To characterize the genetic background of NALT-CLL, we analyzed cytogenetic data and NGS-targeted sequencing for 13 genes in circulating CLL cells (Figure 1B, Table S1). The karyotype was normal in only 2/24 cases (8%) and complex (>3 abnormalities) in 5/24 cases (20%). Half of the patients had trisomy 12 (12/24; 50%); 13q, 17p, and 11q deletions were found in 29%, 8%, and 4% of patients, respectively. A total of 11 patients harbored one mutation (44%), and nine patients carried two to five mutations. TP53 and SF3B1 mutations occurred in 20% (5/25 cases) and 12% (3/25 cases), respectively. Mutations of NOTCH1 pathway components were found in half of the cases (11/25 NOTCH1 and 2/25 FBXW7 mutated cases). Notably, 75% of patients (n = 18/24) had a NOTCH1 abnormality and/or trisomy 12. Similarly, a high frequency of NOTCH1 mutation (34%) and trisomy 12 (29%) has been reported in CLL with specific skin infiltration, while it usually accounts for 5%–10% of patients with CLL at diagnosis and approximately 20% when considering those treated with immunochemotherapy.^{1, 4, 5} This observation raises the question of the role of NOTCH1 mutation and trisomy 12 in extranodal localization of tumor cells.

To gain insight into the molecular mechanism associated with rhinopharyngeal involvement, we screened expression of a set of 70 genes related to cell migration and cell adhesion pathways using a qPCR array in the peripheral CLL cells of patients with NALT (n = 4) compared with no NALT-CLL (n = 4) (Table S2). Genes significantly upregulated with a fold change >2 included the C-C motif chemokine receptor CCR7 (p = 0.05), the C-X motif receptor involved in leukocyte trafficking CXCR3 (p = 0.01) and the chemoattractant chemokine-like factor CKLF. Among the most downregulated genes, we found the gene encoding the hypoxia inducible factor 1 subunit alpha HIF1A, and genes encoding chemokines such as the chemokine (C-C motif) ligand 28 (CCL28) and 3 (CCL3), the interleukine 4 (IL4), the chemokine (C-X motif) ligand (CXCL16), and the tumor necrosis factor (TNF). By targeted qPCR, we confirmed upregulation of CCR7 (p = 0.002), CXCR3 (p = 0.04), and CCR5 (p = 0.03) in the series of 25 NALT-CLL patients compared to 20 age-matched CLL patients without head and neck symptoms (77% with unmutated IGHV, 30% with NOTCH1 mutation and 30% with trisomy 12) (Figure 1C). Upregulation of those targets appeared to be independent of the presence of NOTCH1/trisomy12 aberration or IGHV mutation status, except for CCR5 which was slightly upregulated in the IGHV mutated subgroup (p = 0.04) (Figure S1). In line with these results, immunohistochemistry analysis for five patients with nasal involvement showed strong staining of the CCR7 marker on the membrane of CLL cells infiltrating the mucosa (Figure 1D). The CCR7 receptor is usually expressed on the membrane of CLL cells, and it plays a pivotal role in cell endothelial transmigration within the lymph node in response to binding of CCL19 and CCL21 produced by cells of the microenvironment.⁶ Interestingly, staining for CCL21, the cognate ligand of CCR7, was positive in the high endothelial venules (HEVs) of the nasal mucosa (Figure 1E).

The microenvironment of the rhinopharynx is not a usual niche for CLL cells. Nasal lymphoid tissue forms a unique gateway that plays a central role in induction of local and systemic responses to a wide range of pathogens and allergens. It is composed of T and B cells organized in follicle-associated epithelium, dendritic cells, microfold cells, and macrophages, surrounded by lymphatic vessels and HEVs.⁷ Activated pathogen-recognition receptors in the mucosa induce release of leukocyte-recruiting cytokines and lymphocyte proliferation and differentiation to promote the immune response.⁸ We can therefore hypothesize that localization of CLL cells within the NALT relies on similar mechanisms of cell migration as in lymph nodes, involving interplay between the chemokines produced by the mucosa and matching receptors on CLL cells. In lymph nodes, CCL21 is a potent B-cell chemoattractant expressed by HEVs, nurselike cells (NLCs) and follicular reticular cells in the T-cell zone (FRCs) and plays a pivotal role in driving cell entry and motility in interstitial lymph nodes, favoring their interaction with T cells expressing CD40L.^{6, 9, 10} The presence of CCL21-positive staining in the vessels associated with perivascular distribution of CLL cells in the mucosa strongly supports the hypothesis of either a mechanism of CCL21/CCR7-driven migration in patients with ENT localization, or an enhanced signal of CLL cells retention provided by the CCR7 in the vicinity of the CCL21-expressing HEVs cells. Similarly, it has been shown that expression of CCR7 by CLL cells is significantly higher in patients presenting with lymphadenopathy, suggesting a higher migration capacity and retention within the lymphoid niche.⁶ Finally, it has been demonstrated that NOTCH1 activation or mutation increases expression of CCR7 to promote chemotaxis by suppressing the tumor-suppressor gene DUSP22.¹¹ In our series, NOTCH1 was the most frequently mutated gene, which might enhance migration within the nasal mucosa through upregulation of the CCR7 receptor. However, the absence of significant correlation between CCR7 expression and NOTCH1 mutation suggests that other mechanisms might be involved.

To conclude, we report here cases of specific rhinopharyngeal localization of CLL associated with ENT symptoms. This atypical presentation of CLL is often encountered in patients with progressive disease. It is important to raise awareness among clinicians of the existence of these specific localizations, as their presence should lead to a consideration for faster treatment of CLL, as opposed to delaying it by giving repetitive antibiotic treatment.

由于与治疗相关的体液免疫抑制和/或免疫抑制，慢性淋巴细胞白血病（CLL）患者容易出现感染性并发症，包括耳鼻喉（ENT）感染。然而，尚未描述头部和颈部非淋巴区域的特定 CLL 浸润导致耳鼻喉科症状但与感染无关的情况。 CLL 是一种淋巴器官疾病，但最近的研究报道该疾病累及皮肤和肺部的结外定位。^{1, 2}在这里，我们报告了一系列 CLL 细胞特定定位于鼻咽粘膜的病例，表现为耳鼻喉科感染的症状。为了深入了解这种 CLL 实体的特征，我们回顾性分析了 25 例特定耳鼻喉科受累患者的临床、组织学和分子特征。

所有患者均在血液科进行了确诊 CLL 的随访，他们在 CLL 诊断后中位时间 2.5 年 [1-11 年] 开始出现耳鼻喉科表现。在首次出现耳鼻喉科症状时，所有患者均出现绝对淋巴细胞计数升高（平均为 33 × 109/L [9–117]），并且几乎所有病例均累及淋巴区域，伴有淋巴结肿大和/或脾肿大（ 21 名患者，83%）。耳鼻喉科症状包括慢性咳嗽（44%）、前后流鼻涕（44%）、鼻塞（33%）和咽炎（33%）。这些症状与上呼吸道感染有关，但在抗生素治疗后，这些症状会反复出现并持续存在。进行彻底的耳鼻喉科检查后发现，超过一半的病例（56%）发现粘膜有异常颗粒状，表现为浅褐色病变，增厚、肿胀、橙红色，大部分位于咽后壁。

鉴于经抗生素治疗后耳鼻喉科症状持续存在且无感染迹象，所有患者均接受了鼻或咽粘膜活检（口咽17例，鼻咽5例，扁桃体3例，鼻窦2例）。组织学和免疫化学分析显示具有表达 CD20、CD5 和 CD23 标记物的 CLL 表型的小淋巴细胞浸润。 63% 的病例浸润模式为弥漫性，37% 为血管周围浸润模式（图 1A），这与鼻相关淋巴组织 (NALT-CLL) 中 CLL 细胞的特定受累一致。

就 NALT-CLL 患者的临床结果而言，大多数患者的预后特征较差。大多数病例IGHV未突变（n  = 18/25，72%）。没有检测到IGHV库的偏差，也没有检测到 CDR3 刻板印象。此外，所有病例均根据CLL国际研讨会标准进展（即贫血和/或血小板减少症的发展或恶化、进行性或有症状的脾肿大和/或淋巴结肿大、淋巴细胞倍增时间<6个月）。³ 13 名患者的症状是在一线治疗前确诊的，需要在首次出现耳鼻喉科症状后 2 年内接受治疗。其余 12 名患者在复发时出现耳鼻喉症状。这表明 NALT-CLL 与更进展的疾病相关。有趣的是，全身治疗使所有患者的鼻咽症状完全消退，进一步证实了耳鼻喉科病变与血液病之间的克隆关系。

为了表征 NALT-CLL 的遗传背景，我们分析了循环 CLL 细胞中 13 个基因的细胞遗传学数据和 NGS 靶向测序（图 1B，表 S1）。仅 2/24 例 (8%) 核型正常，5/24 例 (20%) 核型复杂（> 3 个异常）。一半患者患有 12 三体（12/24；50%）；分别有 29%、8% 和 4% 的患者存在 13q、17p 和 11q 缺失。共有 11 名患者携带一种突变（44%），9 名患者携带 2 至 5 种突变。TP53和SF3B1突变发生率分别为 20%（5/25 例）和 12%（3/25 例）。一半病例中发现 NOTCH1 通路成分突变（11/25 NOTCH1和 2/25 FBXW7突变病例）。值得注意的是，75% 的患者 ( n  = 18/24) 存在NOTCH1异常和/或 12 三体性。同样，据报道，在具有特定皮肤的 CLL 中， NOTCH1突变 (34%) 和 12 三体性 (29%)的频率较高浸润，而诊断时通常占 CLL 患者的 5%–10%，而考虑到接受免疫化疗的患者时，这一比例约为 20%。^{1, 4, 5}这一观察结果提出了NOTCH1突变和 12 三体在肿瘤细胞结外定位中的作用问题。

为了深入了解与鼻咽部受累相关的分子机制，我们使用 qPCR 阵列筛选了 NALT 患者 ( n  = 4) 与无NALT 患者的外周 CLL 细胞中一组 70 个与细胞迁移和细胞粘附途径相关的基因的表达。NALT-CLL（n  = 4）（表 S2）。倍数变化显着上调 >2 的基因包括 CC 基序趋化因子受体CCR7 ( p  = 0.05)、参与白细胞运输的 CX 基序受体CXCR3 ( p  = 0.01) 和趋化因子样因子CKLF。在下调幅度最大的基因中，我们发现了编码缺氧诱导因子 1 α 亚基HIF1A 的基因，以及编码趋化因子（CC 基序）配体 28 ( CCL28 )和 3 ( CCL3 )、白细胞介素 4 ( IL4 ) 等趋化因子的基因，趋化因子（CX 基序）配体 ( CXCL16 ) 和肿瘤坏死因子 ( TNF )。通过靶向 qPCR，我们证实 与 20 名年龄匹配的无头颈 CLL 患者相比，25 名 NALT-CLL 患者系列中CCR7 ( p  = 0.002)、CXCR3 ( p  = 0.04) 和CCR5 ( p = 0.03)上调症状（77% 为未突变IGHV，30% 为NOTCH1突变，30% 为 12 三体）（图 1C）。这些靶标的上调似乎与NOTCH1 /trisomy12 畸变或IGHV突变状态的存在无关，但 CCR5 除外，CCR5 在 IGHV 突变亚组中略有上调（p  = 0.04）（图 S1）。与这些结果一致，对 5 名鼻腔受累患者的免疫组织化学分析显示，浸润粘膜的 CLL 细胞膜上的 CCR7 标记物呈强染色（图 1D）。 CCR7 受体通常表达在 CLL 细胞膜上，它在淋巴结内细胞内皮迁移中发挥关键作用，以响应微环境细胞产生的 CCL19 和 CCL21 的结合。⁶有趣的是，CCL21（CCR7 的同源配体）染色在鼻粘膜高内皮小静脉 (HEV) 中呈阳性（图 1E）。

鼻咽部的微环境并不是 CLL 细胞的常见环境。鼻淋巴组织形成了一个独特的门户，在诱导对多种病原体和过敏原的局部和全身反应中发挥着核心作用。它由组织在滤泡相关上皮、树突细胞、微褶皱细胞和巨噬细胞中的 T 细胞和 B 细胞组成，周围环绕着淋巴管和 HEV。⁷粘膜中激活的病原体识别受体会诱导白细胞募集细胞因子的释放以及淋巴细胞的增殖和分化，从而促进免疫反应。⁸因此，我们可以假设 CLL 细胞在 NALT 内的定位依赖于与淋巴结中类似的细胞迁移机制，涉及粘膜产生的趋化因子与 CLL 细胞上的匹配受体之间的相互作用。在淋巴结中，CCL21 是一种有效的 B 细胞趋化剂，由 HEV、护士样细胞 (NLC) 和 T 细胞区 (FRC) 中的滤泡网状细胞表达，在驱动间质淋巴结中的细胞进入和运动方面发挥着关键作用。有利于它们与表达 CD40L 的 T 细胞相互作用。^{6, 9, 10}血管中 CCL21 阳性染色的存在与粘膜中 CLL 细胞的血管周围分布相关，强烈支持以下假设：耳鼻喉科定位患者中 CCL21/CCR7 驱动的迁移机制，或信号增强CCR7 提供的 CLL 细胞保留在表达 CCL21 的 HEV 细胞附近。同样，研究表明，患有淋巴结肿大的患者中，CLL 细胞的 CCR7 表达显着较高，表明其在淋巴微环境中的迁移能力和保留能力较高。⁶最后，已证明 NOTCH1 激活或突变会增加 CCR7 的表达，从而通过抑制肿瘤抑制基因DUSP22来促进趋化性。¹¹在我们的系列中，NOTCH1是最常见的突变基因，它可能通过上调 CCR7 受体来增强鼻粘膜内的迁移。然而，CCR7 表达与 NOTCH1 突变之间缺乏显着相关性，表明可能涉及其他机制。

总之，我们在此报告与耳鼻喉科症状相关的 CLL 特定鼻咽定位病例。 CLL 的这种非典型表现经常出现在疾病进展的患者中。重要的是要提高临床医生对这些特定定位的存在的认识，因为它们的存在应该导致考虑更快地治疗 CLL，而不是通过重复抗生素治疗来延迟治疗。