第一部分 PCDHGB7的异常甲基化及表达作为晚期非小细肺癌免疫标志物探索

研究背景：非小细胞肺癌（non-small cell lung cancer，NSCLC）是全球癌症相关死亡的主要原因之一，免疫检查点抑制剂（immune checkpoint inhibitor，ICIs）的应用显著改变了其治疗格局，但免疫治疗存在低反应率和耐药问题。目前，肿瘤突变负荷（tumor mutation burden, TMB）、PD-L1等生物标志物被用于预测免疫治疗效果，但这些标志物依赖于肿瘤组织活检，存在有创性、检测标准不一致及无法动态监测等缺点。PCDHGB7是原钙粘蛋白家族的重要成员，其在多种肿瘤中的异常甲基化和异常表达现象引起了广泛关注，但其在肺癌中的作用尚未完全明确。

研究方法：我们使用公共数据集（HPA、TCGA、GEO、OncoDB和MEXPRESS等）全面研究了PCDHGB7的表达、预后、生物学功能、甲基化模式及其与免疫浸润和免疫治疗反应的关系。对来自本临床中心的非小细胞肺癌免疫治疗队列进行血浆中PCDHGB7的甲基化和蛋白质水平的检测，以研究其与免疫治疗结果之间的关系。

研究结果：肺腺癌（Lung Adenocarcinoma, LUAD）和肺鳞状细胞癌（Lung Squamous Cell Carcinoma, LUSC）中PCDHGB7表达下调，与肿瘤预后有关。PCDHGB7的甲基化水平在肺癌组织中上调，与PCDHGB7 mRNA水平呈负相关。PCDHGB7与肿瘤突变负荷（Tumor Mutation Burden, TMB）和同源重组缺陷（Homologous Recombination Deficiency, HRD）等基因组标志物呈负相关。PCDHGB7的高表达与M2型巨噬细胞和调节性T细胞（Treg）的浸润呈显著正相关，提示其可能通过影响免疫微环境抑制抗肿瘤免疫反应。在公共免疫治疗队列研究中，PCDHGB7组织表达较高的患者预后较差。本中心临床队列检测显示基线血浆中PCDHGB7高甲基化的患者无进展生存期（progression-free survival，PFS）和总生存期（overall survival，OS）较短，而PCDHCB7甲基化早期减少的患者预后最好。此外，动态监测血浆PCDHGB7甲基化水平可提前揭示疾病进展，其变化早于影像学检查结果约101-175天。血浆PCDHGB7蛋白水平可以预测对免疫检查点抑制剂的反应，并作为PFS的预后标志物。

研究结论：PCDHGB7的表达和甲基化是非小细胞肺癌的预后和免疫标志物。血浆PCDHGB7甲基化和蛋白质水平可作为预测肺癌免疫治疗疗效的生物标志物。

第二部分 PD-1抑制剂联合节拍口服长春瑞滨后线治疗老年晚期非小细胞肺癌患者的疗效、安全性及蛋白标志物探索

研究背景：驱动基因阴性的非小细胞肺癌的治疗仍然面临挑战，特别是在获得了对PD-1/PD-L1抑制剂耐药性的情况下。与常规化疗相比，节拍化疗（metronomic chemotherapy, MCT）指以较小的剂量、高频次地给予化疗药物，其具有更好的安全性、抗肿瘤血管生成作用和免疫调节作用。在这项研究中，我们进行了一项回顾性分析，以评估PD-1抑制剂与节拍口服长春瑞滨（metronomic oral vinorelbine, mOV）联合治疗作为二线及后线治疗转移性非小细胞肺癌患者的疗效和安全性。此外，我们通过血浆蛋白质组学检测确定了该治疗方法相关的蛋白标志物。

研究方法：本研究纳入经组织学或细胞学证实为不可切除的III期和IV期老年非小细胞肺癌患者，并至少接受1种系统性抗肿瘤治疗后进展。患者接受PD-1抑制剂联合mOV（30mg，TIW1，口服）治疗，直至疾病进展或出现无法忍受的副作用。主要终点是无进展生存期（PFS）。10名患者在治疗开始和早期（治疗后1-3个阶段）采集了外周血浆，并使用SOMAscan人血清检测试剂盒进行分析11000种人类蛋白质的表达。

研究结果：从2020年5月到2023年6月，共23名患者进行了纳入研究。中位年龄为70岁（范围：65-85岁），其中18名（78%）为男性。中位随访时间为35.6个月（范围27.2个月-44.13个月），中位无进展生存期为5.63个月（95%CI：4.03-16.5个月）。中位OS为24.8个月（95%CI：13.5个月-NA）。ORR和DCR分别为13%（95%Cl：1.44%-27.6%）和69.6%（95%Cl：49.2%-89.9%）。20名患者（86.9%）出现了任何级别的不良事件，其中4名患者（17.4%）出现了III-IV级不良事件。4名（17.4%）患者发生了免疫相关不良事件（irAE），其中2名（8.7%）G3间质性肺炎患者发生了III-IV级irAE。根据PFS的长度，患者分为short PFS和long PFS组。基线差异蛋白分析鉴定出321种差异蛋白，通路富集结果表明，它们主要富集在胆固醇通路、JAK-STAT信号通路、PI3K-Akt信号通路、PD-1检查点通路和其他通路中。两组患者治疗前后差异蛋白通路富集结果有明显差异。治疗前后的关键差异蛋白分析确定了SH2D2A和HOMER3两种关键蛋白，这两种蛋白质的水平在治疗前long PFS组较低，在治疗后升高，而在short PFS组则呈相反趋势。

研究结论：PD-1抑制剂联合mOV在老年患者后线治疗中具有良好的疗效和安全性。蛋白质生物标志物可以帮助准确识别将从这种治疗方案中受益的患者。

Part І. Exploration of abnormal methylation and expression of PCDHGB7 as an immune marker for advanced non-small cell lung cancer

Background: Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths worldwide, and the use of immune checkpoint inhibitors (ICIs) has significantly changed its treatment landscape. However, immunotherapy has low response rates and drug resistance issues. At present, biomarkers such as tumor mutation burden (TMB) and PD-L1 are used to predict the efficacy of immunotherapy, but these biomarkers rely on tumor tissue biopsy and have disadvantages such as invasiveness, inconsistent detection standards, and inability to monitor dynamically. PCDHGB7 is an important member of the procalcitonin family, and its abnormal methylation and downregulation in various tumors have attracted the attention of researchers. However, its role in lung cancer is not fully understood.

Methods: We conducted a comprehensive analysis of PCDHGB7, focusing on its expression, prognostic significance, biological functions, methylation patterns, and associations with immune infiltration and immunotherapy response. To achieve this, we leveraged publicly datasets, including the Human Protein Atlas (HPA), The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), OncoDB, and MEXPRESS. Additionally, we examined the methylation and protein levels of PCDHGB7 in plasma samples from non-small cell lung cancer (NSCLC) immunotherapy cohorts sourced from our clinical center. This investigation aimed to elucidate the potential correlation between PCDHGB7 and immunotherapy outcomes, thereby providing novel insights into the role of PCDHGB7 in NSCLC immunotherapy.

Results: The downregulation of PCDHGB7 expression in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) is associated with tumor prognosis. PCDHGB7 is positively correlated with suppressive immune cells and negatively correlated with tumor mutation burden (TMB) and homologous recombination deficiency (HRD). The methylation level of PCDHGB7 is upregulated in tumor tissues and negatively correlated with the level of PCDHGB7 mRNA. PCDHGB7 is negatively correlated with genomic markers such as tumor mutation burden (TMB), microsatellite instability (MSI), and homologous recombination deficiency (HRD). The high expression of PCDHGB7 is significantly positively correlated with the infiltration of M2 macrophages and regulatory T cells (Tregs), suggesting that it may suppress anti-tumor immune responses by affecting the immune microenvironment. In immunotherapy cohort studies, patients with high expression of PCDHGB7 tissue have a poorer prognosis. Patients with high baseline plasma levels of PCDHGB7 methylation have shorter progression free survival (PFS) and overall survival (OS), while patients with early reduction of PCDHCB7 methylation have the best prognosis. In addition, dynamic monitoring of plasma PCDHGB7 methylation levels can reveal disease progression in advance, with changes occurring approximately 101-175 days earlier than imaging examination results. The level of plasma PCDHGB7 protein can predict response to immune checkpoint inhibitors and serve as a prognostic marker for PFS.

Conclusions: PCDHGB7 expression and methylation are prognostic and immunological biomarkers in non-small cell lung cancer. Plasma PCDHGB7 methylation and protein levels can be used as novel biomarkers for predicting the efficacy of immunotherapy in lung cancer.

Part Ⅱ. Efficacy, Safety, and Protein Markers of PD-1 Inhibitors Plus Metronomic Oral Vinorelbine in Elderly Metastatic Non-small-cell Lung Cancer Patients

Background: The treatment of non-small cell lung cancer (NSCLC) lacking driver oncogenes remains a formidable challenge, particularly in cases where resistance to programmed cell death protein 1/programmed death-ligand 1 (PD-(L)1) blockade has been acquired. metronomic chemotherapy (MCT), which involves the frequent administration of chemotherapy drugs at reduced doses, offers a more favorable safety profile, anti-tumor angiogenesis, and immune modulatory effects compared to conventional chemotherapy. In this study, we conducted a retrospective analysis to assess the efficacy and safety of combining PD-1 inhibitors with metronomic oral vinorelbine (mOV) in patients with pre-treated metastatic NSCLC. Additionally, we identified protein markers associated with this novel treatment approach through plasma proteomics testing.

Methods: Patients with histologically or cytologically confirmed unresectable old stage III and IV NSCLC and received at least one systematic treatment. Pts received PD-1 inhibitors combined with mOV (30mg, TIW1, day 1-3-5 per week) until disease progression or intolerable side effects. The primary endpoint was progression-free survival (PFS). Peripheral plasma was collected from 10 patients at the beginning and early stages of treatment (1–3 stages after treatment) and were analyzed using the SOMAscan Assay Kit for human serum, which measures the expression of 11000 types of human proteins using highly selective single-stranded modified Slow Off-rate Modified DNA Aptamers (SOMAmer).

Results: From May 2020 to June 2023, 23 pts were evaluated for inclusion in the study. The median age was 70(range: 65-85) with 18(78%) males. Median follow-up was 35.6 months (range 27.2months-44.13months), and the median PFS was 5.63 months (95%Cl: 4.03-16.5 months). The median OS was 24.8 months (95%Cl: (13.5 months-NA). The ORR and DCR were 13% (95%C1: 1.44%-27.6%) and 69.6% (95%Cl: 49.2%-89.9%), respectively. Adverse events (AEs)with any grade were observed in 20(86.9%) pts, of which grade III-IV AEs were observed in 4 (17.4%) pts. Immune-related adverse events (irAEs) occurred in 4 (17.4%) pts, of which grade III-IV irAEs occurred in 2 (8.7%) patients with G3 interstitial pneumonia. According to the length of PFS, patients were divided into a good efficacy group and a poor efficacy group. Baseline differential protein analysis identified 321 differential proteins, and pathway enrichment results showed that they were mainly enriched in the JAK-STAT signaling pathway, AMPK signaling pathway, PI3K Akt signaling pathway, PD-1 checkpoint pathway, and other pathways. The differential protein analysis before and after treatment identified SH2D2A and HOMER3, two proteins involved in T-cell activation. The levels of these two proteins were lower in the good efficacy group before treatment and increased after treatment, while they showed the opposite trend in the poor efficacy group.

Conclusions: The combination of PD-1 inhibitors plus mOV has good efficacy and safety in the postoperative treatment of elderly patients. Protein biomarkers can help accurately identify the patients that will benefit from this treatment regimen.