三阴性乳腺癌新辅助与辅助治疗临床研究及生物标志物探索

第一部分
卡铂联合紫杉类新辅助治疗三阴性乳腺癌生存结果及疗效相关生物标志物探索：一项前瞻性、多中心、队列研究

第1章 卡铂联合紫杉类方案新辅助治疗三阴性乳腺癌生存结果：一项前瞻性、多中心、队列研究

【背景】蒽环联合紫杉类方案是三阴性乳腺癌（triple negative breast cancer, TNBC）新辅助化疗推荐的治疗方案。多项小样本II期临床研究显示卡铂联合紫杉类方案具有与其相似的病理完全缓解率（pathologic complete response, pCR）。目前卡铂联合紫杉类方案新辅助治疗TNBC尚缺乏大样本临床研究及生存数据。

【方法】本研究是一项前瞻性、多中心、队列研究，共在四家医院开展。入选经病理确诊的II-III期TNBC患者，接受卡铂联合紫杉类方案新辅助化疗6个周期：卡铂AUC 4，每2周(q2w)或AUC 5，每3周(q3w)；紫杉类包括紫杉醇（175 mg/m²,q2w或q3w）、白蛋白紫杉醇（220 mg/m²,q3w）或多西他赛（75 mg/m²,q3w）。主要研究终点为无复发生存（recurrence free survival, RFS），次要终点为总生存（overall survival, OS）、pCR率、疗效相关生物标志物研究等。

【结果】2014年1月至2023年12月，共纳入了267例患者，中位年龄为 49岁（范围24~76岁），57.7%（154/267）患者为绝经前。58.4%（156/267）患者临床分期III期。77.1%（206/267）患者淋巴结阳性，临床淋巴结分期cN3患者占17.2%（46/267）。接受紫杉醇治疗患者173例（64.8%），白蛋白紫杉醇78例（29.2%），多西他赛16例（6.0%）。120例患者（44.9%）接受2周方案，147例患者（55.1%）接受3周方案。257例（96.3%）患者完成了至少4个周期的化疗，214例（80.1%）患者完成了至少6个周期。263例患者化疗后接受了乳腺手术治疗，术后pCR率为 40.3%（106/263），微小残留病灶（ypT1mi/1a/1b N0）患者占比13.7%（36/263）。接受白蛋白紫杉醇治疗的患者pCR率显著高于紫杉醇（52.0% vs.35.5%，P=0.02），多变量Logisitc回归分析显示，接受白蛋白紫杉醇治疗和Ki-67≥50%是达到pCR的独立影响因素。中位随访时间为36.0个月，3年RFS和OS分别为 77.9%、87.6%。排除cN3患者后，3年RFS和OS分别为83.1%、91.3%。达到pCR患者比非pCR的患者具有显著更好的RFS和OS（3年RFS 95.5% vs. 68.5%，3年OS 97.7% vs. 81.2%，Log-rank P＜0.001）。2周和3周方案的生存结果相似（3年RFS 78.7% vs. 76.6%，Log-rank P=0.620；3年OS 88.1% vs. 86.8%，Log-rank P=0.548）。经过多变量调整后，新辅助治疗前或治疗后淋巴结分期是RFS的独立影响因素。排除cN3患者后，微小残留病灶患者也具有较好的生存，与pCR患者相比，3年RFS及OS无统计学差异，分别为80.2% vs. 92.3%（Log-rank P=0.060），90.5% vs. 97.4%（Log-rank P=0.247）。残留淋巴结数≥3的患者，3年RFS和OS均差于残留淋巴结数＜3的患者（均Log-rank P＜0.0001）。

【结论】我们大样本研究的结果进一步证实，卡铂联合紫杉类方案新辅助治疗TNBC具有与蒽环联合紫杉方案相当的RFS及OS结果，可作为TNBC患者新辅助“降阶梯”治疗的选择。后续可开展其与免疫治疗联合的临床研究，探索免疫联合化疗的最佳化疗搭档。

第2章 卡铂联合紫杉类方案新辅助治疗三阴性乳腺癌疗效相关的生物标志物研究：基于外周血cfDNA全甲基化组检测的分析

【背景】 卡铂联合紫杉类方案新辅助化疗三阴性乳腺癌（triple negative breast cancer，TNBC）患者具有与蒽环紫杉方案相当的疗效及生存结果，但是目前尚缺乏有效的疗效相关生物标志物。基于游离DNA（cell-free DNA，cfDNA）全甲基化组测序（whole-methylome sequencing，WMS）的生物标志物在乳腺癌新辅助治疗中尚未见报道。

【方法】本研究是基于第1章前瞻性、多中心、队列研究的转化研究。纳入接受卡铂联合紫杉类新辅助化疗的TNBC患者，前瞻性在新辅助治疗前（T1）和新辅助治疗后（T2）收集外周血标本进行外周血cfDNA 低深度WMS检测。获得cfDNA的特征片段染色体拷贝数变异（chromosomal aneuploidy of featured fragments，CAFF）、片段长度特征（fragment size index，FSI）和甲基化密度得分（methylation density score，MD）。探索上述生物标志物与病理完全缓解（pathologic complete response，pCR）以及无复发生存（recurrence free survival，RFS）的相关性。

【结果】2020年9月至2023年5月，收集66例患者共120份外周血标本（64份T1，56份T2），54例患者同时收集到T1及T2标本。患者中位年龄50.5岁，临床分期T3~T4的患者13例（19.7%），临床淋巴结阳性患者53例（80.3%）。63例患者接受手术，33例患者达pCR。中位随访时间16个月，共有10例患者出现疾病复发。T1时CAFF阳性患者43例（67.2%），FSI阳性患者43例（67.2%），MD阳性患者38例（59.4%）。T2时CAFF阳性患者12例（22.2%），FSI阳性患者15例（27.8%），MD阳性患者46例（82.14%）。CAFF及FSI在治疗前后呈下降趋势（P＜0.001），MD呈上升趋势（P=0.009）。病理疗效方面，T1时FSI、CAFF和MD状态与是否达到pCR无明显相关性。T2时，pCR患者中FSI阴性比例高于non-pCR患者（86.2% vs. 59.3%, P=0.034）。CAFF、FSI及MD的动态变化与是否达pCR无显著相关性（P值分别为0.219、0.053、0.297）。基于WMS检测的免疫浸润分析发现T1时CD8+效应记忆T细胞的高免疫浸润评分（P=0.012）、未成熟B细胞的低免疫浸润评分（P=0.017）与达到pCR存在显著相关性。预后方面，T1时MD呈阳性的患者RFS显著差于MD阴性患者（HR=3.74, P=0.033）。T2时CAFF、FSI、MD阳性患者RFS均差于阴性患者，但差异未达统计学意义。

【结论】新辅助治疗后FSI阴性与达到pCR显著相关，基线时MD阳性患者预后较差。本研究结果初步显示基于外周血cfDNA WMS的生物标志物可为TNBC患者接受新辅助卡铂联合紫杉类的治疗提供一定的疗效预测和预后信息，需要在更大规模的队列中以及更长时间的随访进行验证。

第二部分
三阴性乳腺癌新辅助化疗后病理有残留病灶患者辅助化疗真实世界研究及预后标志物探索

第1章 三阴性乳腺癌新辅助化疗后病理有残留病灶患者辅助化疗真实世界研究

【背景】三阴性乳腺癌（triple negative breast cancer, TNBC）新辅助化疗后病理有残留病灶患者预后较差。CREATE-X研究显示针对这类患者卡培他滨辅助治疗能够改善生存，目前已作为指南推荐，但目前国内尚缺乏真实世界数据的报告。本研究旨在报告真实世界里TNBC新辅助化疗后有残留病灶患者辅助治疗的真实情况。

【方法】本研究纳入了2008年7月至2024年12月在中国医学科学院肿瘤医院接受新辅助化疗且术后病理存在残留病灶的TNBC患者。收集患者的临床、病理资料及辅助治疗信息，同时随访获得生存资料。根据术后辅助治疗分为卡培他滨组、静脉化疗组和无化疗组。采用Kaplan-Meier方法绘制生存曲线，Log-Rank检验比较三组间的无病生存期（disease free survival，DFS）以及总生存期（overall survival, OS），多因素Cox比例风险模型用于校正混杂因素。对患者进行Neo-bioscore（NB）评分，并根据残留病灶负荷分为接近病理完全缓解（near-pathological complete response, near-pCR）及高复发风险残留病灶（high-risk residual disease, H-RD）组；near-pCR患者定义为乳腺残留肿瘤直径＜1cm，同时腋窝淋巴结阴性；其余患者为H-RD。根据NB评分以及残留病灶负荷进行分层分析。

【结果】本研究共纳入了340例患者，中位年龄为 47.4岁（范围20~76岁），61.8%（210/340）患者为绝经前。接受含卡培他滨辅助化疗患者120例，接受静脉化疗患者118例，未行辅助化疗患者102例，三组间基线特征基本均衡。卡培他滨组中，63.3%（76/120）患者仅接受卡培他滨治疗，36.7%（44/120）接受了静脉化疗后再口服卡培他滨治疗。静脉化疗组中，39.8%（47/118）患者接受蒽环类联合环磷酰胺（EC）方案，36.4%（43/118）患者接受紫杉类联合卡铂（TCb）方案，12.7%（15/118）患者接受蒽环类联合紫杉类（AT）方案。中位随访时间为37个月，卡培他滨组、静脉化疗组及无化疗组3年DFS分别为81.5%、64.3%、70.5%，3年OS分别为88.4%、81.0%、74.5%。经多因素分析调整后，相比于无化疗组，卡培他滨组能够显著降低疾病复发风险及死亡风险（无化疗vs.卡培他滨：DFS HR=1.92，P=0.032；OS HR=2.51，P=0.026）; 相比于静脉化疗组，卡培他滨组能够显著降低疾病复发风险（静脉化疗vs.卡培他滨：DFS HR=1.84，P=0.037）。分层分析显示，NB≥5的患者中，卡培他滨组的DFS显著优于无化疗组及辅助化疗组，3年DFS分别为73.2%、47.4%、46.5%（卡培他滨组vs.无化疗组Log-rank P=0.043；卡培他滨组vs.静脉化疗组Log-rank P=0.043），卡培他滨组的OS也显著优于无化疗组，3年OS分别为84.0%、53.6%（Log-rank P=0.012）。NB＜5的患者中，3组间DFS及OS未见显著差异。H-RD患者中卡培他滨组DFS及OS均优于无化疗组及静脉化疗组，3年DFS 分别为81.7%、66.5%、61.1%，3年OS分别为 90.3%、70.2%、78.5%（All Log-rank P＜0.05），near-pCR患者中3组间DFS及OS未见显著差异（All Log-rank P＞0.05）。

【结论】本研究展示了TNBC新辅助化疗后病理有残留病灶患者真实世界中辅助治疗选择的多样性，结果显示含卡培他滨的辅助治疗相比于无辅助化疗能够显著降低患者复发风险及死亡风险，特别是NB≥5或者H-RD患者获益明显。

第2章 三阴性乳腺癌新辅助化疗后病理有残留病灶患者预后标志物研究

【背景】三阴性乳腺癌（triple negative breast cancer ,TNBC）新辅助化疗后未达病理完全缓解（non pathologic complete response, non-pCR）患者预后不良，但并非所有患者均预后不佳，残留肿瘤负荷（residual cancer burden，RCB）可提供一定预后信息，但仍缺乏分子层面的有效标志物。本研究旨在从蛋白组学层面分析TNBC新辅助治疗后残留病灶患者预后的生物标志物。 

【方法】基于第二部分第1章的研究基础，将TNBC新辅助化疗后残留病灶患者根据随访生存结果分为短期复发组（无复发生存≤3年）及未复发组（无复发生存＞3年），并进行组间基线特征匹配。60例患者残留肿瘤标本进行基于液相色谱-质谱联用技术的全蛋白组分析，复发组与未复发组之间进行差异蛋白筛选分析、通路富集分析、蛋白互作网络分析、免疫浸润分析。基于蛋白组分析结果，在免疫组化水平验证关键差异蛋白的表达。

【结果】60例患者术后残留肿瘤样本接受检测，复发组30例，未复发组30例，两组间基线特征均衡。复发组对比未复发组共鉴定出49种蛋白上调，237种蛋白下调。GO富集分析显示下调蛋白多富集在白细胞介导的免疫反应、T细胞激活和线粒体代谢相关通路。KEGG富集分析显示下调蛋白富集在T细胞受体信号通路、破骨细胞分化相关通路。两种通路富集分析均显示，复发患者相比于未复发患者免疫通路相关蛋白尤其是T细胞通路显著下调。蛋白互作网络分析筛选出25个关键蛋白，关键蛋白中多个为免疫相关蛋白CD1C、CD247、CD2、CD300A、CD3D、CD40、CD68、CD84、GZMB、CD20、CD150。基于25个关键蛋白的生存分析结果显示，CD40和CD68下调对患者的生存影响最显著。免疫浸润分析显示复发患者免疫相关细胞表达下调，包括CD4+效应T细胞、树突状细胞、巨噬细胞、巨噬细胞M2、浆细胞样树突状细胞等在复发组表达下降。结合关键差异蛋白及免疫浸润分析结果，在免疫组化验证CD4、CD40、CD68在复发组及未复发组间的表达，结果显示复发组表达水平显著下降，复发组中肿瘤浸润淋巴细胞表达水平也显著低于未复发组（中位10% vs. 20%，P＜0.001）。

【结论】本研究发现，在TNBC新辅助治疗后残留病灶患者中，免疫相关蛋白尤其是T细胞通路下调与患者短期复发密切相关。免疫组化结果进一步证实CD4、CD40、CD68在复发组表达下降。初步证实了免疫相关蛋白表达下降是TNBC残留病灶患者预后的重要影响因素，为后续深入研究和临床实践提供了有价值的参考和方向。

Clinical Research and Biomarker Exploration of Neoadjuvant and Adjuvant Therapy for Triple-Negative Breast Cancer

PART 1  

Survival outcomes and efficacy related biomarkers exploration of carboplatin plus taxanes as neoadjuvant chemotherapy in triple negative breast cancer: A prospective, multicenter, cohort study

Chapter 1   Survival outcomes of carboplatin plus taxanes as neoadjuvant chemotherapy in triple negative breast cancer：A prospective, multicenter, cohort study

【Background】The anthracyclines plus taxane is widely acknowledged as a recommended regimen for neoadjuvant chemotherapy (NAC) in the treatment of triple negative breast cancer (TNBC). Several small-scale phase II studies indicate that the carboplatin plus taxanes regimen achieves a comparable pathologic complete response (pCR) in TNBC. However, there is still a lack of large-sample studies and survival data on the carboplatin plus taxanes as NAC in TNBC.

【Methods】 This is a prospective multi-center cohort study conducted in four hospitals of China. Eligible patients had pathologically confirmed stage II - III TNBC were enrolled to receive NAC of carboplatin plus taxanes: carboplatin AUC 5 every 3 weeks(q3w) or AUC 4 every 2 weeks(q2w); taxanes included paclitaxel (175mg/m²,q2w or q3w), nab-paclitaxel (220 mg/m²,q2w), or docetaxel (75mg/m², q3w). The primary endpoint assessed in the study was recurrence free survival (RFS). Additionally, secondary endpoints encompassed overall survival (OS), the rate of pCR, and exploratory biomarker analyses.

【Results】Between January 2014 and December 2023, a total of 267 patients were enrolled in the study with a median age of 49 year(range 24~76 years). 57.7% of the patients (154/267) were premenopausal. 58.4% of the patients (156/267) were stage III disease. 206 (77.1%) patients were lymph node positive and clinical node stage cN3 accounted for 17.2% (46/267). A total of 173 patients (64.8%) received paclitaxel, 78 (29.2%) received nab-paclitaxel, and 16 (6.0%) received docetaxel. 120 patients(44.9%) received a biweekly regimen, and 147 (55.1%) with tri-weekly regimen. Of all patients, 257 (96.3%) completed more than 4 cycles, and 214 (80.1%) completed 6 cycles. 263 patients underwent the surgery and 40.3% patients (106/263) achieved pCR. Patients with minimal residual disease (ypT1mi/1a/1b N0) accounted for 13.7% (36/263). The pCR rate was significantly higher in patients receiving nab-paclitaxel than in those receiving paclitaxel (52.0% vs. 35.5%, P = 0.02). Multivariable logistic regression analysis revealed that nab-paclitaxel treatment and and Ki-67≥50% were independent predictors of pCR. After a median follow up duration of 36.0 months, the 3-year RFS and OS were 77.9%, 87.6%, respectively. Excluding patients with cN3, 3-year RFS and OS were 83.1%,91.3%, respectively. Patients who achieved pCR had a significant better RFS and OS than those non-pCR (3 year RFS 95.5% vs. 68.5%，3 year OS 97.7% vs. 81.2%，Log-rank P＜0.001). There were no significant differences in survival outcomes between the biweekly and the triweekly group (3-year RFS 78.7% vs. 76.6%, Log-rank P=0.62; 3-year OS 88.1% vs.86.8%, Log-rank P=0.548). After multivariate adjustment, the N stage before and after NAC was an independent influencing factor for RFS. After excluding cN3 patients, survival of patients with minimal residual disease (ypT1mi/1a/1b N0) are comparable to those pCR, 3-year RFS and OS were 80.2% vs. 92.3%(Log-rank P=0.060), 90.5% vs. 97.4% (Log-rank P=0.247), respectively. Patients with residual node number≥3 had a poor 3-year RFS and OS compared to those number＜3(All Log-rank P＜0.0001).

【Conclusion】The findings of our large-sample study further confirmed that neoadjuvant carboplatin plus taxanes regimen in TNBC offers comparable RFS and OS outcomes to anthracycline-taxanes regimen and can be a "de-escalation" treatment choice. Future clinical trials should investigate the efficacy of combining carboplatin with taxanes and immunotherapy, with the aim of identifying the optimal chemotherapeutic partner with immunotherapy.

Chapter 2  Biomarker study of carboplatin plus taxanes as neoadjuvant chemotherapy in triple negative breast cancer: Analysis based on the whole methylome sequencing of cfDNA in peripheral blood

【Background】Carboplatin plus taxanes neoadjuvant chemotherapy for triple negative breast cancer (TNBC) patients has comparable efficacy and survival outcomes to anthracycline-taxanes, but currently, there is a lack of effective biomarkers. The biomarkers based on cell-free DNA (cfDNA) of whole-methylome sequencing (WMS) has not been reported yet in breast cancer neoadjuvant therapy.

【Methods】The study is a translational research based on the prospective, multicenter, cohort study in Chapter One. Patients with TNBC who received neoadjuvant chemotherapy with taxanes plus carboplatin were included. Plasma samples were prospectively collected at baseline(T1) and end of NAC (T2). Chromosomal aneuploidy of featured fragments (CAFF), fragment size index (FSI) and methylation density score (MD) of cfDNA were detected with WMS. Explore the correlation of the above mentioned biomarkers with pathologic complete response(pCR) and recurrence free survival(RFS).

【Results】From September 2020 to May 2023, a total of 66 patients with 120 plasma samples (64 samples at T1, 56 samples at T2) were included in WMS analysis, 54 patients had samples collected at both T1 and T2. The median age of the patients was 50.5 years. 13 patients (19.7%) with clinical stage T3-T4 and 53 patients (80.3%) were lymph node positive. 63 patients underwent surgery, and 33 patients achieved pCR. The median follow-up time was 16 months, and a total of 10 patients experienced disease recurrence. At the timepoint of T1, 43 (67.2%) of the patients were CAFF positive, 43 (67.2%) were FSI positive, and 38 (59.4%) were MD positive. At the timepoint of T2, 12 (22.2%) were CAFF positive, 15 (27.8%) were FSI positive, and 46 (82.14%) were MD positive. CAFF and FSI showed a downward trend after treatment (P<0.001), while MD showed an upward trend (P=0.009). In terms of pathological efficacy, there was no significant difference in FSI, CAFF and MD status between the pCR and non-pCR groups at T1. The proportion of patients with FSI negative at T2 was significantly higher in pCR group compared to non-pCR(86.2% vs. 59.3%, P=0.034) at T2. There was no significant correlation between the changes in CAFF, FSI and MD status and whether pCR was achieved (P values were 0.219, 0.053 and 0.297, respectively). Immune infiltration analysis revealed that a high immune infiltration score of CD8+ effector memory T cells at T1 (P=0.012) and a low immune infiltration score of immature B cells (P=0.017) were significantly associated with achieving pCR. In terms of prognosis, patients with MD positive at T1 was significantly associated with poor RFS compared to MD negative (HR=3.74, P=0.033). While patients positive for CAFF, FSI or MD at T2 also had worse RFS than negative, but the differences were not statistically significant.

【Conclusion】 FSI negative after neoadjuvant therapy was significantly associated with achieving pCR. Baseline MD status was related to prognosis, and patients with positive MD had a poorer prognosis. Biomarkers based on cfDNA WMS could provide predictive and prognostic information for TNBC patients receiving neoadjuvant carboplatin plus taxane regimen, warranting further investigation.

PART 2

Adjuvant chemotherapy for patients with residual disease after neoadjuvant chemotherapy in triple-negative breast cancer：A real-world study and prognostic marker exploration

Chapter 1  Adjuvant chemotherapy for patients with residual disease after neoadjuvant chemotherapy in triple-negative breast cancer：A real-world study

【Background】Triple-negative breast cancer (TNBC) patients exhibiting residual disease post-neoadjuvant chemotherapy are associated with a poor prognosis. The CREATE-X study indicates that capecitabine adjuvant therapy benefits these patients, particularly the TNBC subgroup, and is now guideline-recommended. However, there's a lack of real-world data in China. This study aims to present the real-world adjuvant treatment status of TNBC patients with residual disease after neoadjuvant therapy.

【Methods】This study enrolled TNBC patients with residual disease post-neoadjuvant chemotherapy after surgery at the Cancer Hospital, Chinese Academy of Medical Sciences, from July 2008 and December 2024. Clinical, pathological data, adjuvant therapy information were collected, and survival data was obtained via follow up. Patients were categorized into capecitabine, intravenous chemotherapy, and no chemotherapy groups based on adjuvant treatment. The Kaplan-Meier method and the Log-Rank test was used to compare disease free survival(DFS) and overall survival(OS) among the three groups. A multivariate Cox proportional hazards model was used to adjust for confounding factors. The Neo-bioscore (NB) was assessed in all patients, and according to the residual disease burden, we divided them into near-pCR (breast tumor diameter < 1cm and axillary lymph node negative) and high-risk residual disease(H-RD) groups. Stratified analysis was done based on NB scores and residual disease burden.

【Results】A total of 340 patients were included in this study, with a median age of 47.4 years (range 20-76 years), 61.8% of patients were premenopausal. There were 120 patients who received adjuvant chemotherapy containing capecitabine, 118 patients who received intravenous chemotherapy, and 102 patients who did not receive adjuvant chemotherapy. The baseline characteristics were generally balanced among the three groups. In the capecitabine group, 63.3% (76/120) of patients received only capecitabine, and 36.7% (44/120) received intravenous chemotherapy followed by capecitabine. In the intravenous chemotherapy group, 39.8% (47/118) received the anthracycline plus cyclophosphamide (EC) regimen, 36.4% (43/118) the taxane plus carboplatin(TCb) regimen, and 11.0% (13/118) the anthracycline plus taxane(AT) regimen. The median follow-up time was 37 months. The 3-year DFS for the capecitabine group, intravenous chemotherapy group, and no chemotherapy group were 81.5%, 64.3%, and 70.5%, respectively, and the 3-year OS were 88.4%, 81.0%, and 74.5%, respectively. After adjustment by multivariate analysis, compared with the no chemotherapy group, the capecitabine group could significantly reduce the risk of disease recurrence and death (No chemotherapy vs. Capecitabine: DFS HR=1.92, P=0.032; OS HR=2.51, P=0.026); compared with the intravenous chemotherapy group, the capecitabine group could significantly reduce the risk of disease recurrence (Intravenous chemotherapy vs. Capecitabine: DFS HR=1.84, P=0.037). Stratified analysis for patients with NB≥5 showed the capecitabine group had better DFS than the no chemotherapy and adjuvant chemotherapy groups, with 3 - year DFS rates of 73.2%, 47.4%, and 46.5%, respectively(Capecitabine group vs. No chemotherapy group, Log-rank P=0.043; Capecitabine group vs. Intravenous chemotherapy group, Log-rank P=0.043), and the OS was also significantly better than that of the no chemotherapy group, with 3-year OS of 84.0% and 53.6%, respectively (Log-rank P=0.012). In patients with NB<5, there was no significant difference in DFS and OS among the three groups. In H-RD patients, the DFS and OS of the capecitabine group were better than those of the no chemotherapy group and the intravenous chemotherapy group, with 3-year DFS of 81.7%, 66.5%, and 61.1%, respectively, and 3-year OS of 90.3%, 70.2%, and 78.5%, respectively (All Log-rank P<0.05), while in near-pCR patients, there was no significant difference in DFS and OS among the three groups (All Log-rank P>0.05).

【Conclusion】The study presents the real world adjuvant therapy options for TNBC patients with residual disease after neoadjuvant chemotherapy. Adjuvant capecitabine significantly reduces recurrence and mortality risks compared to no adjuvant chemotherapy, especially in patients with NB≥5 or H-RD.

Chapter 2   Prognostic biomarker research in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy

【Background】Triple negative breast cancer(TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor prognosis. However, not all such patients have a bad outcome. Residual cancer burden (RCB) can provide some prognostic information, but there is still a lack of effective molecular markers. This study is aimed at exploring the prognostic biomarkers in TNBC patients with residual disease after neoadjuvant therapy from the perspective of proteomics.

【Methods】Based on the research in Chapter 1 of this section, TNBC patients with residual disease after neoadjuvant chemotherapy were divided into a short-term recurrence group (recurrence free survival≤3 years) and a non-recurrence group (recurrence free survival> 3 years), and baseline characteristics between the two groups were matched. Sixty residual tumor samples from patients were selected for whole proteome analysis using Liquid Chromatography-Mass Spectrometry (LC-MS). Differential protein screening, pathway enrichment analysis, protein - protein interaction network analysis, and immune infiltration analysis were performed between the two groups. Finally, the expression of differential proteins was validated at the immunohistochemical(IHC) level.

【Results】A total of 60 postoperative residual tumor samples were tested, with 30 in the recurrence group and 30 in the non-recurrence group, and baseline characteristics were balanced between the two groups. A total of 49 up-regulated and 237 down-regulated proteins were identified when comparing the recurrence and non-recurrence groups. GO enrichment analysis indicated that the down-regulated proteins were mainly enriched in pathways related to leukocyte-mediated immune response, T cell activation, and mitochondrial metabolism. KEGG enrichment analysis showed that the down-regulated proteins were enriched in the T cell receptor signaling pathway and osteoclast differentiation pathway. Both pathway enrichment analyses revealed that immune-related proteins, especially those in the T cell pathway, were significantly down-regulated in recurrent patients compared to non-recurrent patients. Protein-protein interaction network analysis identified 25 key proteins, several of which are immune-related, including CD1C, CD247, CD2, CD300A, CD3D, CD40, CD68, CD84, GZMB, CD20, and CD150. Survival analysis based on these 25 key proteins showed that down-regulation of CD68 and CD40 most significantly affected patient survival. Immune infiltration analysis revealed down-regulated immune cell-related expression in recurrent patients compared to non-recurrent patients, with decreased expression of CD4+ effector T cells, dendritic cells, macrophages, M2 macrophages, naive B cells and plasmacytoid dendritic cells in the recurrence group. Based on the analysis of key differential proteins and immune infiltration, IHC was used to verify the expression of CD4, CD40, and CD68 in recurrence and non-recurrence groups. Results showed that the recurrence group had significantly lower expression levels. The tumor-infiltrating lymphocytes in the recurrence group were also significantly lower than in the non-recurrence group (median 10% vs. 20%, P<0.001).

【Conclusion】The study found that in patients with residual disease after neoadjuvant therapy for TNBC, the downregulation of immune-related proteins, particularly in the T cell pathway, is closely associated with short term recurrence. Immunohistochemistry confirmed that CD4, CD40, and CD68 are downregulated in the recurrent group. Our study preliminarily confirms that the downregulation of immune-related proteins is an important prognostic factor for TNBC patients with residual disease, offering valuable insights for future research and clinical practice.