第一部分 HER2表达转移性乳腺癌RC48-ADC治疗疗效与预后生物标志物的探索

摘要

背景：维迪西妥单抗（Disitamab vedotin, RC48）是一种新型抗体偶联药物（ADC），由抗人类表皮因子生长受体2（HER2）单克隆抗体与细胞毒剂单甲基奥瑞他汀E（MMAE）通过可裂解连接子偶联而成，在既往治疗失败的 HER2 阳性和 HER2 低表达转移性乳腺癌（MBC）患者中展现出一定的疗效。本研究旨在通过对HER2表达转移性乳腺癌的蛋白组学进行探索性分析，筛选出疗效预测的生物标志物。

方法：收集患者治疗前的福尔马林固定石蜡包埋（FFPE）组织样本，并提取肿瘤样本的（磷酸化）蛋白质进行反相蛋白阵列（RPPA）检测。分析RPPA定量检测HER2表达与HER2免疫组化（IHC）分类的相关性，并筛选出临床疗效相关的生物标志物，包括客观缓解率（ORR）和无进展生存期（PFS）。最后，利用最小绝对收缩和选择算子（Lasso）回归，筛选目的蛋白构建RC48的疗效预测模型，并进行模型验证。

结果：共有33名HER2阳性和HER2低表达MBC患者接受了RPPA检测。Spearman相关性分析显示，RPPA和IHC分析HER2表达具有良好的相关性（r=0.56，P=0.02）。基于HER2 RPPA检测的截断值0.38，高HER2表达（＞0.38）患者的中位PFS显著优于低HER2表达患者（9.7个月 vs 4.7个月，P＜0.001）。通过对总体蛋白组进行无监督聚类区分RC48治疗敏感组和非敏感组，后筛选出两组的差异表达蛋白质（P < 0.05, |Log2 FC| > 0.8）共8个。对差异蛋白进行KEGG和KSEA富集分析，发现PI3K/AKT/mTOR通路激活患者对RC48的敏感性更高。利用Lasso回归建立了新型ADC药物疗效预测模型（ADC-RS），并在验证集中进行验证，受试者工作特征（ROC）的曲线下面积（AUC）为0.875。

结论：RPPA检测HER2表达与新型ADC类药物疗效显著相关。本研究首次确定了HER2低表达患者ADC疗效预测的最佳HER2截断值，并基于蛋白组学构建了ADC药物疗效预测模型，这为根据分子标志物区分优势人群提供了依据，并有望为个体化方案的制定提供支持。

第二部分 RC48治疗PAM通路异常活化的HER2表达的经治转移性乳腺癌有效性和安全性的II期临床研究

摘要

背景：维迪西妥单抗（Disitamab vedotin；RC48）是我国首个原创靶向HER2抗体偶联药物，在人表皮因子受体2（HER2）阳性和低表达转移性乳腺癌（MBC）表现出良好的疗效和安全性。本研究旨在探讨RC48治疗PI3k/Akt/mTOR（PAM）通路异常激活的HER2表达的经治转移性乳腺癌患者的疗效和安全性

方法：该研究是一项前瞻性、单臂II期临床试验，于2022年9月至2023年12月期间招募了经组织或血液学基因检测伴有PAM通路异常激活的HER2阳性和HER2低表达经治转移性乳腺癌患者，接受RC48（2mg/kg，每2周一次，静脉滴注）治疗。主要研究终点为客观缓解率（ORR），次要终点为疾病控制率（DCR）、无进展生存（PFS）、总生存期（OS）和安全性。该研究在ClinicalTrials.gov上进行了注册，注册号为NCT05331326。

结果：本研究共纳入62名经组织学或外周血基因检测证实伴有PAM通路异常激活的HER2表达转移性乳腺癌患者，其中HER2阳性MBC患者26例，HER2低表达MBC患者36例。中位随访时间为13.2个月，RC48治疗HER2表达MBC的ORR为34.4%（21/61），HER2阳性组和HER2低表达组分别为34.6%（9/26）和34.3%（12/31）。RC48治疗的中位PFS为3.5个月（95%置信区间[CI]：2.4-4.6个月），HER2阳性和HER2低表达患者的中位PFS分别为4.5个月（95%CI：2.9-6.1个月）和3.4个月（95%CI：2.8-4.0个月）。两组之间PFS无统计学差异（风险比[HR]=0.67；95%CI，0.37−1.22；P=0.18）。安全性方面，常见3级及以上治疗相关不良事件包括神经毒性（11.3%），中性粒细胞下降（8.1%），天门冬氨酸氨基转移酶（AST）升高（3.2%）和白细胞下降（3.2%），未出现因治疗药物导致死亡情况。

结论：对于PI3K/Akt/mTOR通路异常活化的经治HER2阳性和低表达转移性乳腺癌人群，RC48显示出一定的疗效（ORR为34.6%）且安全性良好，不良反应可控。RC48可能是PAM通路异常活化HER2表达转移性乳腺癌患者的治疗选择之一。

第三部分 HER2表达状态对CDK4/6抑制剂联合内分泌治疗HR阳性转移性乳腺癌的疗效的影响

摘要

背景：目前细胞周期蛋白依赖性激酶4/6（CDK4/6）抑制剂联合传统的内分泌治疗是激素受体（HR）阳性人表皮生长因子受体2（HER2）阴性转移性乳腺癌（MBC）的标准一线治疗方案。然而，对于HER2低表达和HER2零表达患者的CDK4/6抑制剂联合治疗获益尚不清楚。本研究旨在评估HER2低表达和HER2零表达对HR阳性MBC患者CDK4/6抑制剂联合内分泌治疗疗效的影响。

方法：本研究纳入了1,186名PALOMA-2（NCT01740427）和PALOMA-3（NCT01942135）随机对照临床试验中的HR阳性、HER2阴性MBC患者，分析了HER2低表达和HER2零表达患者的无进展生存期（PFS）。 研究纳入2016年4月至2019年11月中国医学科学院肿瘤医院前瞻性队列233例HR阳性、HER2阴性MBC患者作为外部验证。在PALOMA-2中，患者被随机分配接受哌柏西利或安慰剂联合来曲唑一线治疗HR阳性MBC。PALOMA-3研究中，患者被随机分配接受二线及以上哌柏西利或安慰剂联合氟维司群治疗。外部验证队列患者接受一线哌柏西利联合内分泌治疗或单独内分泌治疗。本研究主要终点是由研究者评估的PFS。

结果：在PALOMA-2研究的666名MBC患者中，有153名HER2零表达患者和513名HER2低表达患者。在HER2零表达患者中，哌柏西利-来曲唑组与安慰剂-来曲唑组的PFS无显著差异（风险比=0.79，95%置信区间[CI] 0.48–1.30，p=0.34）。在HER2低表达患者中，哌柏西利-来曲唑组的PFS显著优于安慰剂-来曲唑组（风险比=0.52，95% CI 0.41–0.66，p<0.0001）。PALOMA-3研究分析了520名MBC患者。在153名HER2零表达患者中，哌柏西利-氟维司群组的PFS显著优于安慰剂-氟维司群组（风险比=0.54，95% CI 0.30–0.95，p=0.034）。同样在367名HER2低表达患者中，哌柏西利-氟维司群较安慰剂-氟维司群显著改善了PFS（风险比=0.39，95% CI 0.28–0.54，p<0.0001）。外部验证队列分析示，在HER2零表达患者中，哌柏西利联合治疗组与单独内分泌治疗组的PFS无显著差异（风险比=0.80，95% CI 0.30–2.08，p = 0.66）。在HER2低表达患者中，哌柏西利联合治疗组的PFS显著优于单纯内分泌治疗组（风险比=0.24，95% CI 0.13–0.46，p < 0.0001）。

结论：CDK4/6抑制剂联合内分泌治疗显著改善了HER2低表达患者的预后，本研究强调了HER2低表达状态对转移性乳腺癌CDK4/6抑制剂治疗的重要性，未来仍需要前瞻性研究进一步证实。

PART 1 Proteomic biomarkers predictive of response and survival following RC48-ADC treatment for HER2-expressing metastatic breast cancer

Abstract

Background: Disitamab vedotin (RC48), a novel antibody-drug conjugate (ADC) , demonstrated promising efficacy in pretreated HER2-positive and HER2-low metastatic breast cancer (MBC). In this study, we performed an exploratory analysis of the proteomics of HER2-expressing in MBC to identify predictive biomarkers for treatment response and survival outcomes.

Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples were collected prior to treatment initiation and (phosphorylated) proteins from tumor samples were extracted for reverse-phase protein array (RPPA) testing. Correlation analysis between quantitative HER2 expressing detected by RPPA and immunohistochemistry (IHC) classification was conducted. Subsequently, biomarkers associated with clinical efficacy, including objective response rate (ORR) and progression-free survival (PFS), were identified. A least absolute shrinkage and selection operator (lasso) regression was employed to screen target proteins and construct a predictive model for ADC efficacy, followed by model validation.

Results: A total of 33 patients with HER2-positive and HER2-low MBC underwent RPPA testing. Spearman correlation analysis revealed a positive correlation between RPPA and IHC test of HER2 expression (r=0.56, P=0.02). Using a cutoff value of 0.38 for the HER2 assay, the median PFS was significantly longer in patients with high HER2 expressing than in those with low expression (9.7 vs 4.7 months, P<0.001). A total of 8 differentially expression proteins (P < 0.05, |Log2 FC| > 0.8) were identified in both RC48-sensitive and resistant groups through unsupervised clustering of the overall proteome. KEGG and KSEA enrichment analysis revealed heightened RC48 sensitivity in patients with abnormal activation of the PI3K/AKT/mTOR pathway. A novel ADC efficacy prediction model (ADC-RS) was established using lasso regression and validated in an independent cohort, achieving an area under the receiver operating characteristic curve of 0.875.

Conclusion: RPPA-detected HER2 expressing significantly correlated with novel ADC efficacy. This study identified the optimal HER2 cut-off value for predicting ADC efficacy in HER2 low MBC patients and constructed a predictive model based on proteomics, suggesting a molecularly stratified approach for ADC treatment and supporting personalized treatment strategies.

PART2 Disitamab vedotin (RC48) for HER2-expressing pretreated metastatic breast cancer with abnormal activation of PAM pathway: A prospective phase II study

Abstract

Background: Disitamab vedotin (RC48) is the first domestically developed antibody-drug conjugate targeting HER2, demonstrating promising efficacy and safety in HER2-positive and HER2-low metastatic breast cancer (MBC). This study aimed to explore the efficacy and safety of RC48 in HER2-expressing pretreated MBC patients with abnormal activation of the PI3k/Akt/mTOR (PAM) pathway.

Methods: This prospective, single-arm phase II clinical trial recruited HER2-positive and HER2 low MBC patients who had progressed on at least one line of systemic chemotherapy with confirmed PAM pathway activation from September 2022 to December 2023. The primary endpoint was objective response rate (ORR), and secondary endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and safety. The study was registered on ClinicalTrials.gov with the registration number NCT05331326.

Results: A total of 62 patients with confirmed PAM pathway activation were enrolled, including 26 patients with HER2-positive MBC and 36 with HER2 low MBC. The median follow-up time was 13.2 months. The ORR for HER2-expressing MBC was 34.4% (21/61), with rates of 34.6% (9/26) in the HER2-positive patients and 34.3% (12/31) in the HER2 low-expression group. The median PFS with RC48 was 3.5 months (95%CI: 2.4-4.6 months), with 4.5 months (95%CI [confidence interval]: 2.9-6.1 months) in HER2-positive and 3.4 months (95%CI: 2.8-4.0 months) in HER2 low patients. There was no statistically significant difference in PFS between the two groups (hazard ratio=0.67; 95%CI, 0.37−1.22; P=0.18). Regarding safety, the most common grade 3 or higher treatment-related adverse events included neuropathy (11.3%), neutropenia (8.1%), aspartate aminotransferase elevation (3.2%), and leukopenia (3.2%), with no treatment-related deaths.

Conclusions: RC48 demonstrated moderate efficacy with a manageable safety profile in HER2-positive and HER2-low pretreated MBC patients with abnormal activation of the PI3k/Akt/mTOR pathway. RC48 may be a therapeutic option for patients with HER2-expressing MBC with PAM pathway abnormal activation.

PART3 The impact of HER2 expression status on the efficacy of CDK4/6 inhibitors combined with endocrine therapy in HR-positive metastatic breast cancer

Abstract

Background: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors combined with traditional endocrine therapy (ET) have become the recommended first-line treatment for hormone receptor (HR)-positive and HER2-negative metastatic breast cancer (MBC). However, the benefits of adding CDK4/6 inhibitors to ET in HER2-low and HER2-zero subgroups remain unclear. We aimed to evaluate the impact of HER2 expressing on the efficacy of CDK4/6 inhibitors combined with ET in patients with HR-positive MBC.

Methods: This secondary analysis assessed progression-free survival (PFS) among HER2-low and HER2-zero patients enrolled in the double-blind, placebo-controlled randomized clinical trials PALOMA-2 and PALOMA-3. The study included 1,186 HER2-negative, HR-positive female patients across 17 countries enrolled between February 2013 and August 2014. Additionally, an external validation cohort of 233 HR-positive, HER2-negative MBC patients from Cancer Hospital, Chinese Academy of Medical Sciences, enrolled between April 2016 and November 2019, was included in the study. In the PALOMA-2 trial, patients were randomly assigned to receive either palbociclib or placebo, in combination with letrozole in the first-line treatment for HR-positive MBC. Patients in the PALOMA-3 study, who had progression or relapse during previous ET, were randomly allocated to receive either palbociclib plus fulvestrant or placebo plus fulvestrant. The external validation cohort patients received either first-line palbociclib combined with endocrine therapy or endocrine therapy alone. The primary endpoint was investigator-assessed PFS. The two trials are registered with ClinicalTrials.gov, number NCT01740427 and NCT01942135.

Results: Of the 666 patients with MBC from the PALOMA-2 study, there were 153 HER2-zero and 513 HER2-low patients. In the HER2-zero population, no significant difference in PFS was observed between the palbociclib-letrozole and placebo-letrozole groups (hazard ratio=0.79, 95% confidence interval [CI] 0.48–1.30, p=0.34). In the HER2-low population, palbociclib-letrozole demonstrated a significantly lower risk of PFS than placebo-letrozole group (hazard ratio=0.52, 95% CI 0.41–0.66, p<0.0001). The PALOMA-3 study analysed 520 patients with MBC. Within the 153 HER2-zero patients, the palbociclib-fulvestrant group showed a significantly longer PFS than the placebo-fulvestrant group (hazard ratio=0.54, 95% CI 0.30–0.95, p=0.034). Among the 367 HER2-low patients, palbociclib-fulvestrant improved PFS (hazard ratio=0.39, 95% CI 0.28–0.54, p<0.0001).  The results of the external validation cohort showed no significant difference in PFS between the palbociclib group and the endocrine therapy alone group among HER2-zero patients (hazard ratio = 0.80, 95% CI 0.30–2.08, p = 0.66). However, among HER2-low patients, the palbociclib group exhibited significantly better PFS compared to the endocrine therapy alone group (hazard ratio = 0.24, 95% CI 0.13–0.46, p < 0.0001).

Conclusions: The combination of a CDK4/6 inhibitor with ET significantly improved PFS in HER2-low expression patients. The results of this study highlight the importance of integrating HER2 status in tailoring personalized treatment strategies for HR-positive MBC. Further work is needed to validate these findings.