第一部分 原发性肺腺癌患者外周血TERT阳性白细胞检测方法的建立及其初步临床应用

目的：本研究旨在建立一种基于端粒酶逆转录酶（Telomerase Reverse Transcriptase, TERT）活性的外周血TERT阳性白细胞亚群的检测方法，用于评估恶性肿瘤患者外周血中白细胞的增殖潜能和免疫衰老状态，并将其初步应用于早期肺腺癌患者。

方法：本研究采用改造后的重组I型单纯疱疹病毒（HSVI-hTERTp-eGFP），仅在高TERT启动子转录活性的细胞中复制，启动绿色荧光蛋白（Green Fluorescence Protein, GFP）表达，通过识别GFP，实现对TERT阳性白细胞亚群的检测，从而评估机体的免疫增殖潜能。分别将100、500和1000个经病毒感染后表达GFP的Jurkat细胞，加到2.5 × 10⁶个人外周血白细胞中，以检测模拟样品的回收率、重复性、线性回归系数等指标。检测肺结节患者（n = 5）外周血TERT阳性白细胞百分比（设置3个平行组），通过计算组内变异系数进一步评估技术可靠性。通过衰老相关β-半乳糖苷酶（Senescence Associated- β-galactosidase, SA-β-gal）染色，验证免疫衰老与TERT阳性白细胞百分比的相关性，并通过流式细胞术对外周血TERT阳性白细胞的分群情况进行分析。利用该技术对肺腺癌患者（n = 80）和良性肺结节患者（n = 80）外周血TERT阳性白细胞百分比进行检测，分析TERT阳性细胞百分比与患者临床信息的相关性。最后，对早期肺腺癌患者应用胸腺肽进行术后辅助治疗前后外周血TERT阳性白细胞百分比的变化进行分析。

结果：含有100、500和1000个Jurkat细胞的模拟样本平均检出阳性细胞数分别为80.3、448.2和920.6个，回收率分别为 80.3%、89.6%和92.1%。批间变异系数分别为11.3%、7.2%和4.4%，批内变异系数均在10%以内，临床样本的组内变异系数也均小于10%。以上结果说明该技术具有较高的准确度和良好的稳定性。此外，SA-β-gal染色阳性率与TERT阳性细胞百分比呈负相关（r = −0.58，p < 0.05），提示TERT阳性白细胞百分比与免疫衰老相关。外周血免疫细胞分群显示，TERT阳性白细胞主要分布在粒细胞和单核细胞中。在80例良性肺结节患者中，外周血TERT阳性白细胞百分比随着年龄增长而下降（r = −0.61，p < 0.0001）。早期肺腺癌患者的外周血TERT阳性白细胞百分比显著低于良性肺结节患者（p < 0.0001），而与非浸润性肺腺癌患者相比，浸润性肺腺癌患者外周血TERT阳性白细胞百分比显著更低（p < 0.001）。在应用胸腺肽进行术后辅助治疗后，肺腺癌患者外周血TERT阳性白细胞比例呈上升趋势，但未达到统计学意义。

结论：本研究成功建立并验证了一种肿瘤患者外周血TERT阳性白细胞亚群的检测方法，并进行了初步的临床应用。肺腺癌患者外周血TERT阳性白细胞百分比显著低于良性肺结节患者，存在免疫细胞增殖能力下降的现象，且在浸润性肺腺癌患者中表现更显著，而在应用胸腺肽进行术后辅助治疗后，早期肺腺癌患者外周血TERT阳性白细胞百分比呈上升趋势。这可能与年龄相关的免疫衰老有关，为理解浸润性肺腺癌患者较差的预后提供了一个新视角。

第二部分 乳腺癌患者HER2阳性循环肿瘤细胞及外周血TERT阳性白细胞检测的临床应用研究

目的:在乳腺癌（Breast Cancer, BC）疾病进展过程中，人表皮生长因子受体2（Human Epidermal Growth Factor Receptor 2, HER2）表达呈动态变化。循环肿瘤细胞（Circulating Tumor Cell, CTC）作为一种实时监测HER2表达的手段，具有潜在的临床应用价值。本研究旨在评估乳腺癌患者原发肿瘤灶和CTC之间HER2表达的一致性，并分析外周血TERT阳性白细胞百分比与CTC及患者临床信息的相关性。

方法:本研究使用已建立的基于TERT活性的CTC检测技术（TERT-based CTC Detection, TBCD），结合HER2抗体，检测乳腺癌患者外周血中的CTC和HER2阳性CTC （HER2+ CTC）的数量。同时，利用建立的外周血TERT阳性白细胞检测技术分析患者外周血的免疫增殖状态。

结果：在32.6%的乳腺癌患者观察到原发肿瘤灶和CTC之间HER2状态不一致的情况（Kappa值 = 0.325，p = 0.03）。在原发肿瘤灶组织学HER2阴性的癌症患者中，HER2+ CTC的检出率为32.1%（9/28）。TERT阳性白细胞百分比与HER2+ CTC数量、CTC数量、年龄、肿瘤大小及Ki67指数均呈负向单调递减趋势，但均未达到统计学意义。

结论：本研究建立的HER2+ CTC检测技术可成功检测到肿瘤患者HER2+ CTC，并证实乳腺癌患者的外周血肿瘤原发灶组织学HER2表达情况与CTC的HER2状态不尽一致，突显了检测HER2+ CTC的临床意义。然而，外周血TERT阳性白细胞百分比与乳腺癌患者CTC数量及临床信息之间未见明确相关性，后续应进一步扩大样本量，深入探究外周血免疫状态与乳腺癌疾病的相关性。

Part 1: Establishment and Preliminary Clinical Application of Detection Method for TERT Positive White Blood Cells in Peripheral Blood of Patients with Primary Lung Adenocarcinoma

Objective: This study aims to establish a detection method for peripheral blood TERT positive white blood cell subsets based on telomerase reverse transcriptase (TERT) activity, to evaluate the proliferation potential and immunosenescence status of white blood cells in patients with malignant tumors, and to conduct preliminary clinical applications in early lung adenocarcinoma patients.

Methods:This study used a modified recombinant type I herpes simplex virus (HSVI-hTERTp-eGFP), which can replicate only in cells with high TERT promoter transcriptional activity and activate the expression of green fluorescence protein (GFP). By recognizing GFP, the detection of TERT positive white blood cell subsets can be achieved, thereby evaluating the immune proliferation potential of the body. We added 100, 500, and 1000 Jurkat positive cells expressing GFP after infection with the HSVI-hTERTp-eGFP virus to 2.5 × 10⁶ human peripheral blood leukocytes to prepare simulated samples for testing recovery rate, repeatability, linear regression coefficient, and other indicators. Peripheral blood samples were collected from patients with pulmonary nodules (n = 5) to detect the percentage of TERT positive white blood cells (3 parallel groups were set up), and the reliability of the technique was further evaluated by calculating the coefficient of variation within each group. By staining with senescence associated- β-galactosidase (SA-β-gal), the correlation between immune aging and the percentage of TERT positive white blood cells was verified, and the grouping of peripheral blood TERT positive white blood cells was analyzed by flow cytometry. Using this detection technology, the percentage of TERT positive white blood cells in peripheral blood of patients with lung adenocarcinoma (n = 80) and patients with benign pulmonary nodules (n = 80) was detected, and the correlation between TERT positive cell percentage and patient clinical information was analyzed. Finally, the percentage of TERT positive white blood cells in peripheral blood of early lung adenocarcinoma patients before and after adjuvant therapy with thymosin was detected and analyzed.

Results: The average number of positive cells detected in simulated samples containing 100, 500, and 1000 Jurkat cells were 80.3, 448.2, and 920.6, respectively, with recovery rates of 80.3%, 89.6%, and 92.1%, respectively. The inter batch coefficients of variation were 11.3%, 7.2%, and 4.4%, while the intra batch coefficients of variation were all within 10%. The intra group coefficients of variation for clinical samples were also less than 10%. The above results indicate that the technology has high accuracy and good stability. In addition, the positive rate of SA-β-gal staining was negatively correlated with the percentage of TERT positive cells (r = −0.58，p < 0.05), indicating that the percentage of TERT positive white blood cells is associated with immunosenescence. Peripheral blood immune cell clustering shows that TERT positive white blood cells are mainly distributed in granulocytes and monocytes. In 80 patients with benign pulmonary nodules, the percentage of TERT positive white blood cells in peripheral blood decreased with age (r = −0.61，p < 0.0001). The percentage of TERT positive white blood cells in peripheral blood of patients with early lung adenocarcinoma was significantly lower than that of patients with benign lung nodules (p < 0.0001), while compared with non-invasive lung adenocarcinoma patients, the percentage of TERT positive white blood cells in peripheral blood of patients with invasive lung adenocarcinoma was significantly lower (p < 0.001). After using thymosin for postoperative adjuvant therapy, the proportion of TERT positive white blood cells in peripheral blood of lung adenocarcinoma patients showed an upward trend, but did not reach statistical significance. 

Conclusion:This study successfully established and validated a detection method for TERT positive white blood cell subsets in peripheral blood of cancer patients, and conducted preliminary clinical applications. Research has found that the percentage of TERT positive white blood cells in peripheral blood of patients with lung adenocarcinoma is significantly lower than that of patients with benign pulmonary nodules, indicating a decrease in immune cell proliferation ability in lung adenocarcinoma patients, which is more significant in invasive lung adenocarcinoma patients. After using thymosin for postoperative adjuvant therapy, the percentage of TERT positive white blood cells in peripheral blood of early lung adenocarcinoma patients shows an upward trend. This may be related to age-related immunosenescence, providing a new perspective for understanding the poor prognosis of patients with invasive lung adenocarcinoma.

Part 2: Clinical Application of HER2 Positive Circulating Tumor Cells and TERT Positive Leukocytes in Peripheral Blood of breast cancer Patients

Background：During the progression of breast cancer (BC), the expression of human epidermal growth factor receptor 2 (HER2) showed dynamic changes. Circulating tumor cells (CTCs) have potential clinical application value as a means of real-time monitoring of HER2 expression. The purpose of this study was to evaluate the consistency of HER2 expression between primary tumor focus and CTC in breast cancer patients, and to analyze the correlation between the percentage of TERT positive leukocytes in peripheral blood, CTC and clinical information of patients.

Methods：In this study, the established TERT-based CTC detection (TBCD), combined with HER2 antibody, was used to detect the number of CTC and HER2+CTC in the peripheral blood of breast cancer patients. At the same time, the established peripheral blood TERT positive white blood cell detection technology was used to analyze the immune proliferation status of patients' peripheral blood. 

Results：Discordance in HER2 status between primary tumor and CTC was observed in 32.6% of patients (Kappa value = 0.325, p = 0.03). In patients with histologically HER2-negative primary tumor tissue, the detection rate of HER2+CTC was 32.1% (9/28). The percentage of TERT positive white blood cells showed a negative monotonic decreasing trend with HER2+ CTC count, CTC count, age, tumor size, and Ki67 index, but all of these results did not reach statistical significance.

Conclusions：The HER2+CTC detection technology established in this study can successfully detect HER2+CTC in tumor patients, and confirm that the histological HER2 expression in primary tumor lesions of breast cancer patients' peripheral blood is not consistent with the HER2 status of CTC, highlighting the clinical significance of detecting HER2+CTC. However, there is no clear correlation between the percentage of TERT positive white blood cells in peripheral blood and the number of CTC and clinical information in breast cancer patients. In the future, the sample size should be further expanded to further explore the correlation between the immune status of peripheral blood and breast cancer disease.