题目：甲状腺乳头状癌淋巴结转移个体化治疗和肿瘤微环境特征

中文摘要：

第一部分：甲状腺乳头状癌淋巴结转移个体化治疗的初步研究

研究背景

甲状腺乳头状癌（PTC）近年发病率迅速增长，其特点是淋巴结转移（LNM）的发生率非常高，手术是处理LNM首选且最有效的治疗方式，目前各大指南对侧颈淋巴结清扫（LND）的范围尚未达到共识，仍存在很大争议。本研究旨在探究cN1期PTC患者侧颈LND的手术范围，探索如何在进行最大限度清扫、减少遗漏和复发的同时，减小手术创伤、降低术后并发症、提高患者生活质量，减少无效医疗。

研究方法

本前瞻性研究纳入了自2015年10月至2020年1月于中国医学科学院肿瘤医院头颈外科经同一主诊医师手术的550名PTC患者。对于术前超声考虑有Ⅵ区LNM的患者，经术前穿刺细胞学检查或术中冰冻病理学检查证实后，在原有甲状腺领式切口的基础上直接完成侧颈Ⅲ、Ⅳ区LND；对于术前超声考虑有Ⅲ或/和Ⅳ区LNM的患者，取3-6枚Ⅲ区可疑/肿大淋巴结（包括肩胛舌骨肌上淋巴结）送冰冻病理学检查，如发现存在有LNM则延长切口至胸锁乳突肌后缘完成侧颈Ⅱ-Ⅳ区LND，否则仅完成侧颈Ⅲ、Ⅳ区LND；对于术前超声考虑有Ⅱ（包括Ⅲ或/和Ⅳ）区LNM的患者，经术前穿刺细胞学检查或术中冰冻病理学检查证实后，直接完成Ⅱ-Ⅴ区LND。

研究结果

1.146例术前超声考虑仅有Ⅵ区LNM的患者中，术后病理证实有75例（51.4%）存在Ⅲ、Ⅳ区隐匿性LNM，且Ⅵ区LNM数目与侧颈LNM数目呈线性正相关，Ⅵ区LNM数目≥3是预测Ⅲ、Ⅳ区隐匿性转移的最佳界值，年龄＜55岁、肿瘤大小≥20mm以及Ⅵ区LNM数量与Ⅲ、Ⅳ区隐匿性LNM显著相关，并且Ⅵ区LNM数目≥3是Ⅲ、Ⅳ区隐匿性转移的独立危险因素。

2.术前超声考虑有Ⅲ或/和Ⅳ区LNM的患者中，90例患者因术中冰冻病理提示Ⅲ区淋巴结未见转移而仅行Ⅲ、Ⅳ区LND在随访期间均未出现术野外的复发，317例患者因术中冰冻病理提示Ⅲ区淋巴结存在转移而行Ⅱ-Ⅳ区LND，术后病理证实有146例（46.1%）存在有Ⅱ区隐匿性LNM，Ⅲ区LNM数目≥2是预测侧颈Ⅱ区隐匿性转移的最佳界值，肿瘤甲状腺背膜外侵犯以及Ⅲ区LNM数量与Ⅱ区隐匿性LNM相关。

3.97例术前超声考虑有Ⅱ（包括Ⅲ或/和Ⅳ）区LNM的患者中，术后病理证实有19例（20.0%）存在Ⅴ区隐匿性LNM，且Ⅴ区隐匿性LNM仅与患者性别和是否合并有甲状腺炎相关。

4.围手术期并发症的发生与手术范围未见统计学相关。术后出现伤口周围长期麻木、发紧等不适以及因严重瘢痕增生影响生活质量的患者比例随清扫范围增大而增加，但未达到明显统计学差异。

研究结论

1.依靠超声评估和淋巴结转移规律精准的术前预测，有利于根据患者特征制定个体化的手术方案；

2.规划精准的清扫范围可以有效清除隐匿性LNM以避免治疗不足，同时避免因过度治疗而导致的患者术后生活质量下降；

3.结合甲状腺癌区域淋巴结的转移规律和术前超声检查区域淋巴结受累解剖亚区的数目（n），建议cN1期PTC侧颈LND的范围为n+1/2个解剖亚区。

第二部分：甲状腺乳头状癌淋巴结转移与耗竭性肿瘤免疫微环境的关系

研究背景

甲状腺乳头状癌（PTC）虽总体预后较好，但淋巴结转移（LNM）的发生很常见，目前诊疗过程中外科医生规划手术方案的主要依据是敏感性有限的超声和CT，对于隐匿性LNM的遗漏可能导致手术范围不足而出现转移淋巴结的残留/复发。此外对于因肿瘤广泛而失去外科手术机会或反复复发的难治性PTC，目前还没有公认有效的挽救治疗方案。本研究旨在探究与PTC患者发生LNM和肿瘤进展相关的肿瘤微环境（TME）的特点，期望为分子诊疗手段的补充提供新思路和线索，减少由于治疗不足导致的复发，填补对于无法手术患者补救治疗措施的空白，为复发难治性PTC患者的免疫治疗提供理论依据。

研究方法

本研究共纳入40例于中国医学科学院肿瘤医院头颈外科行手术治疗的PTC患者，通过HE染色、免疫组化染色、组织间液多因子检测及流式细胞染色等技术探索存在有LNM的PTC肿瘤组织中浸润的免疫细胞亚群和功能的变化。同时纳入448例从TCGA公共数据库中获取的PTC样本的转录组学数据和临床数据进行统计分析从而验证TME对LNM的发生可能存在的作用机制。本研究中的所有数据应用FlowJo、SPSS、GraphPad Prism和R软件进行分析并作图。   

研究结果

1.免疫组化染色结果显示发生LNM的患者TME中浸润有更多的CD45⁺的免疫细胞，多因子检测发现，与未发生LNM的患者相比，发生LNM的患者的肿瘤组织细胞间液中存在有更高水平的具有促进肿瘤迁移和侵袭的IL-6和CCL5，而抑制性免疫细胞分子IL-8和IL-10水平具有升高的倾向，但未达到统计学的差异（p=0.100，0.084）。

2.进一步采用流式细胞染色技术对肿瘤组织浸润的免疫细胞类型分析发现，LNM组患者的肿瘤组织中如下细胞群体的浸润显著增高：CD3⁺T细胞，CD3⁺CD8⁺T细胞，CD3⁺CD8⁺PD-1⁺T细胞以及CD3⁺CD8⁺PD-1^hiTIM-3⁺T细胞。其中，PD-1⁺CD8⁺T细胞中的PD-1^hiTIM-3⁺T细胞的比例升高与肿瘤甲状腺背膜外侵犯相关，CD45⁺细胞、CD3⁺T细胞、CD3⁺CD8⁺T细胞和PD-1⁺CD8⁺T细胞的浸润数量与肿瘤直径及合并有甲状腺炎呈正相关，与合并有良性结节呈负相关。合并有LNM的患者肿瘤中的CTLA-4阳性的CD4⁺T细胞升高，并且Treg细胞浸润数量与LNM显著正相关。

3.从TCGA公共数据库中获取的样本转录组数据分析发现，合并LNM的病例样本中存在有更多的负性免疫检查点分子，同时高表达有多种具有抑制免疫效应的细胞因子相关基因；GO分析和KEGG分析发现，合并LNM的肿瘤组织中存在多条抑制性免疫通路的显著上调；肿瘤中PD-1⁺CD8⁺T细胞的富集程度与LNM、肿瘤分期以及病理亚型相关，且与Treg细胞的浸润比例呈线性正相关。PD-1⁺CD8⁺T细胞高富集组的样本同样存在多种具有抑制免疫效应的相关基因的特异性高表达以及多条抑制性免疫通路的显著上调。

研究结论

1.PTC合并有LNM与不合并有LNM的肿瘤相比展现出不同的TME，包括有更多的免疫浸润以及有更多的抑制性免疫细胞因子和趋化因子表达；

2.存在有LNM的患者的肿瘤组织中浸润有更多的耗竭性的PD-1⁺CD8⁺T细胞，同时耗竭性PD-1⁺CD8⁺T细胞的浸润还与肿瘤进展相关，此外CD4⁺T细胞表面CTLA-4的表达以及Treg细胞的浸润也与LNM的发生相关；

3.利用公共数据库数据的分析再次验证了临床标本结果，同时还发现PTC患者的LNM可能是由于多种抑制性免疫通路的上调而导致的；

4.上述结果可能对PTC患者术前对隐匿性LNM辅助诊断的分子标志物的探索以及对复发难治性PTC患者挽救免疫治疗措施的补充提供新线索。

PART I: The Preliminary Study on Personalized Extent of Neck Dissection in Papillary Thyroid Carcinoma

Background

The incidence of papillary thyroid carcinoma (PTC), which is prone to lymph node metastasis (LNM), has increased rapidly in recent years. Surgery is the first and the most effective treatment method for LNM. There is much controversy on the extent of lymph node dissection (LND) for PTC patients with LNM according to major guidelines. This study aims to explore the surgical extent of lateral LND in patients with cN1 PTC, and to explore how to maximize dissection to reduce omissions and recurrences, while reducing trauma caused by surgery or postoperative complications, improving patients' quality of life, and reducing ineffective medical treatment.

Methods

This prospective study included 550 PTC patients who were operated by the same attending physician at the Department of Head and Neck Surgery, Cancer Hospital of the Chinese Academy of Medical Sciences from October 2015 to January 2020. For patients who were suspected to have LNM in level VI by preoperative ultrasound, after being confirmed by preoperative fine-needle aspiration or intraoperative frozen section, LND of level III and IV was directly performed based on the original thyroid collar incision. For patients who were considered to have LNM in level III or/and IV by preoperative ultrasound, 3-6 suspicious/enlarged lymph nodes in level III (including supra-omohyoid lymph nodes) were taken for frozen section. If LNM was found, the incision was extended along the skin striae to the posterior edge of the sternocleidomastoid muscle to complete LND of level II-IV, otherwise, only LND of level III and IV was completed. For patients who were considered to have LNM in level II (including III or/and IV) in preoperative ultrasound, after being confirmed by preoperative fine-needle aspiration or intraoperative frozen section, LND of level II-V was completed directly.

Results

1. Among the 146 patients who were suspected to have LNM in level VI by preoperative ultrasound, 75 patients (51.4%) were confirmed to have occult LNM in level III and IV, and the number of LNM in level III and IV has positive linear correlation with the number of LNM in level VI. The number of LNM in level VI≥3 was the best predictor of occult metastasis in level III and IV. Age＜55 years old, tumor size≥20 mm, and the number of LNM in level VI were significantly related to occult LNM in level III and IV, and LNM≥3 in level VI were the independent risk factor of occult LNM in level III and IV.

2. Among the patients who were considered to have LNM in level III or/and IV preoperatively, 90 patients performed LND of level III and IV as no LNM was found in level III intraoperatively, and no recurrence occurred outside the surgical field during the follow-up period. Meanwhile, of 317 patients who underwent LND of level II-IV as LNM was found in level III intraoperatively, 146 patients (46.1%) were confirmed to have occult LNM in level II. The number of LNM in level III≥2 was the best predictor of occult metastasis in level II. Extrathyroidal extension, and the number of LNM in level III were related to occult LNM in level II.

3. Among the 97 patients who were considered to have LNM in level II (including III or/and IV) in preoperative ultrasound, 19 patients (20.0%) were confirmed to have occult LNM in level V. Gender and thyroiditis were related to occult LNM in level V.

4. The occurrence of perioperative complications was not related to the extent of surgery. The proportion of patients who experienced discomfort such as long-term numbness and tightness around the wound after surgery, as well as scars that seriously affected their quality of life increased as the extent of dissection enlarged, however no statistically significant was found.

Conclusion

1. Accurate preoperative prediction based on ultrasound assessment and lymph node metastasis patterns is conducive to planning an individualized and precise surgical scope based on patient characteristics.

2. Formulating a precise dissection extent could effectively remove occult LNM to avoid under-treatment while avoiding a decrease in the patient’s postoperative quality of life due to over-treatment.

3. Combining with the metastasis pattern of regional lymph nodes in PTC and the number (n) of involved anatomical subregions of lymph nodes in the preoperative ultrasound, it is recommended that the scope of lateral LND for cN1 PTC should be n+1/2 anatomical subregions.

PART II: Papillary Thyroid Carcinoma with Immune Exhaustion is Prone to Lymph Node Metastasis

Background

Although papillary thyroid carcinoma (PTC) has an overall good prognosis, it is prone to lymph node metastasis (LNM). Surgeons usually plan the scope of surgery mainly based on ultrasound and CT, which have limited sensitivity. Occult LNM might be missed and lead to LNM residual/recurrence as insufficient surgical extent. In addition, there is currently no recognized effective salvage treatment for refractory PTC that has lost the chance of surgery due to extensive tumors or relapsed overtimes. This study aims to explore the characteristics of tumor microenvironment (TME) related to LNM and tumor progression in PTC patients, hoping to provide new ideas and clues for the supplementation of molecular diagnostic and therapeutic methods, to reduce recurrence due to insufficient treatment, fill the gap of salvage treatments for inoperable patients, and provide theoretical basis of immunotherapy for refractory patients.

Methods

This study included a total of 40 PTC patients who underwent thyroid surgery at the Department of Head and Neck Surgery, Cancer Hospital of the Chinese Academy of Medical Sciences. HE staining, immunohistochemical staining, interstitial fluid multi-cytokines detection, and flow cytometry were used to explore the changes of subpopulations and functions of immune cells infiltrating in PTC tumor tissues with LNM. While, transcriptomic and clinical data of 448 PTC samples were obtained from the TCGA database for statistical analysis to verify the possible mechanism of TME on the occurrence of LNM. All data in this study were analyzed and graphed using FlowJo, SPSS, GraphPad Prism, and R software.

Results

1.  Immunohistochemical staining showed that patients with LNM had more CD45⁺ immune cells infiltrating in TME. Multi-cytokines detection found that there were more IL-6 and CCL5, which promote tumor migration and invasion, presented in intercellular fluids in the tumor tissue of patients with LNM. And more IL-8 and IL-10, which were suppressive immune cytokines, could be found, though there was no statistically significant (p=0.100, 0.084).

2. Further exploration of subpopulations of immune cells infiltrating in tumor tissues by flow cytometry showed that the infiltration of CD3⁺T cells, CD3⁺CD8⁺T cells, CD3⁺ CD8⁺PD-1⁺ T cells, and CD3⁺CD8⁺PD-1^hiTIM-3⁺T cells were related to LNM, and the proportion of PD-1^hiTIM-3⁺T cells among PD-1⁺CD8⁺T cells was related to the extrathyroidal extension. Moreover, the amount of CD45⁺ cells, CD3⁺T cells, CD3⁺CD8⁺T cells, and CD3⁺CD8⁺PD-1⁺ T cells was associated with tumor size and thyroiditis, and negatively related to benign nodules. PTC patients with LNM also presented more CTLA-4 positive CD4⁺T cells and the infiltration of Treg was significantly positively correlated to LNM.

3. Analysis of the transcriptome data obtained from TCGA showed that there were more negative immune checkpoints in LNM patients and a variety of suppressive-cytokine-related genes were highly expressed. The results also found that multiple inhibitory immune pathways were significantly up-regulated in tumor tissues of LNM patients. In addition, the enrichment of PD-1⁺CD8⁺T cells in tumors was related to LNM, tumor stage, and pathological subtype, and the infiltration of Treg cells was linearly positively related to PD-1⁺CD8⁺T cells. Patients in the PD-1⁺CD8⁺T cell high expression group also had specific high expression of a variety of related genes with suppressive immune effects, as well as significant upregulation of multiple suppressive immune pathways.

Conclusions

1. PTC tumors with LNM exhibited a different TME compared with tumors without LNM, including greater infiltration of leukocytes and more expression of immunosuppressive cytokines and chemokines.

2. Patients with LNM had more exhausted PD-1⁺CD8⁺T cells infiltrating in the tumor tissue, while the infiltration of exhausted PD-1⁺CD8⁺T cells was related to tumor progression. The expression of CTLA-4 on CD4⁺T cells and the infiltration of Treg cells were also related to LNM.

3. After using the public database to re-verify the results from clinical specimens, it was found that LNM in PTC might be caused by the upregulation of multiple suppressive immune pathways.

4. The above results may provide new clues for exploring molecular diagnosis methods for occult LNM in PTC before surgery and salvage immunotherapy for relapsed and refractory PTC patients.