Human epidermal growth factor receptor 2(HER2) status is a crucial predictive factor for prognostic assessment and targeted therapy selection, which may be influenced by intratumor heterogeneity and molecular divergence between the primary site and different metastases. Therefore, we performed a prospective study to confirm the concordance of HER2 amplification in circulating tumor DNA(ctDNA) with primary tumor tissue and verified its clinical implications. A total of 105 breast cancer patients were enrolled, and dynamic monitoring of HER2 copy numbers in ctDNA was conducted in 31 participants during the treatment. Totally 186 plasma samples were prospectively obtained and blinded to test HER2 copy numbers in ctDNA based on low-coverage whole genome sequencing(WGS) by next-generation sequencing(NGS). Comparing HER2 copy numbers in ctDNA collected before the initiation of next line of anticancer treatment with primary tumor tissue, the concordant rate of HER2 amplification was 86.5%(χ2 = 52.901, p < 0.001), with a positive and negative predictive value of 94.9% and 80.7%, respectively. Histopathologically positive, high-level amplification of HER2 copy numbers in the baseline was significantly correlated with best objective response during the anticancer therapy(p = 0.010). Moreover, HER2 copy numbers fluctuated with HER2-targeted therapeutic response, and the patients with a constantly positive level after 6 weeks of treatment appeared to suffer from significantly reduced progression free survival(p < 0.001). HER2 amplification in ctDNA, with a concordance rate of over 80% with primary tumors, may be a predictive index for prognostic evaluation and therapeutic response monitoring in a noninvasive, repeatable and practical method for breast cancer patients.

Antibody-drug conjugates (ADCs) are a transformative approach in breast cancer therapy, offering targeted treatment with reduced toxicity by selectively delivering cytotoxic agents to cancer cells. While ADCs like trastuzumab emtansine (T-DM1), trastuzumab deruxtecan (T-DXd), and sacituzumab govitecan have shown significant efficacy, resistance mechanisms such as antigen loss, impaired internalization, and efflux of cytotoxic payloads challenge their effectiveness. This review discusses these resistance mechanisms and explores advanced strategies to overcome them, including innovations in linker chemistry, multi-antigen targeting, and biomarker-driven personalization. Additionally, therapeutic sequencing - determining the optimal order of ADCs with other treatments such as chemotherapy, endocrine therapy, and immunotherapy - is examined as a crucial approach to maximize ADC efficacy and manage resistance. Evidence-based sequencing strategies, particularly for human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancer (TNBC), are supported by clinical trials demonstrating the benefits of ADCs in both early-stage and metastatic settings. The potential of combination therapies, such as ADCs with immune checkpoint inhibitors (ICIs), further highlights the evolving landscape of breast cancer treatment. As ADC technology advances, personalized approaches integrating biomarkers and optimized sequencing protocols offer promising avenues to enhance treatment outcomes and combat resistance in breast cancer.

Purpose: We used targeted capture sequencing to analyze TP53‐mutated circulating tumor DNA (ctDNA) in metastatic breast cancer patients and to determine whether TP53 mutation has predictive value for anti‐human epidermal growth factor receptor 2 (HER2) treatment for in HER2 amplification‐positive patients (HER2+) and HER2 mutation‐positive, amplification‐negative (HER2−/mut) patients.Patients and Methods: TP53 mutation features were analyzed in the Geneplus cohort (n = 1184). The MSK‐BREAST cohort was used to explore the value of TP53 mutation in predicting anti‐HER‐2 antibody efficacy. Sequencing of ctDNA in phase Ib, phase Ic, phase II clinical trials of pyrotinib (HER2+ patients), and an investigator‐initiated phase II study of pyrotinib (HER2−/mut patients) were performed to analyze the relationships between TP53 mutation and prognosis for HER2 TKIs. The MSK‐BREAST cohort, MutHER, and SUMMIT cohort were used for verification.Results: TP53 mutations were detected in 53.1% (629/1184) of patients in the Geneplus cohort. The TP53 mutation rate was higher in HR‐negative (p < 0.001) and HER2 amplification‐positive (p = 0.015) patients. Among patients receiving anti‐HER2 antibody therapy, those whose tumors carried TP53 mutations had a shorter PFS (p = 0.004). However, the value of TP53 mutation in predicting HER2 TKI response was inconsistent. In HER2+ patients, no difference in PFS was observed among patients with different TP53 statuses in the combined analysis of the pyrotinib phase Ib, phase Ic, and phase II clinical trials (p = 1.00) or in the MSK‐BREAST cohort (p = 0.62). In HER2−/mut patients, TP53 mutation‐positive patients exhibited a trend toward worse prognosis with anti‐HER2 TKI treatment than TP53‐wild‐type patients in our investigator‐initiated phase II study (p = 0.15), and this trend was confirmed in the combined analysis of the MutHER and SUMMIT cohorts (p = 0.01).Conclusions: TP53 mutation can be used to identify biomarkers of anti‐HER2 antibody drug resistance in HER2+ patients and HER2 TKI resistance in HER2−/mut patients.

Everolimus, an inhibitor of mammalian target of rapamycin (mTOR), has been shown to increase the efficacy of endocrine therapies in hormone receptor (HR)-positive metastatic breast cancer. However, because breast cancer is a highly heterogeneous disease, the responses of different patients to everolimus may vary. Therefore, we performed this study to better select patients who will benefit most from or be resistant to everolimus. Patients with HR-positive breast cancer who were treated with everolimus at the Cancer Hospital, Chinese Academy of Medical Sciences from February 2014 to March 2017 were enrolled in the present study. Mutations in ctDNA were assayed in 1021 tumor-related genes via gene panel target capture-based next-generation sequencing. In total, 120 patients with metastatic breast cancer who were treated with everolimus were enrolled in the present study. The median progression-free survival (PFS) of all patients was 5.1 months (95% confidence interval [CI] 3.9–6.3 months). No difference in survival was observed between patients who received endocrine drugs used in previous treatment regimens and patients who did not receive these drugs (median PFS 5.2 and 5.1 months, respectively, p > 0.05). Additionally, we did not find any difference in outcomes between patients who had primary resistance to previously used endocrine drugs and patients who had nonprimary resistance to previous treatments (p > 0.05). Multivariate analysis showed that < 3 metastatic sites, < 2 lines of previous endocrine therapy, < 2 lines of previous chemotherapy, and treatment with everolimus combined with fulvestrant were associated with improved survival (p < 0.05). Sixteen patients underwent ctDNA analysis before everolimus treatment. The frequency of PIK3CA gene mutations was 62.5%, and H1047R was the most frequently detected mutation. Patients with the PIK3CA/H1047R mutation had longer PFS than patients with wild-type or other mutant forms of PIK3CA, and the median PFS in these two groups of patients was 8.8 and 4.1 months, respectively (p < 0.05). Our data suggest that patients who receive more lines of chemotherapy or endocrine therapy are less likely to benefit from everolimus. For everolimus combination therapy, we can even select endocrine drugs that gave rise to primary resistance in previous treatments. Additionally, the PIK3CA/H1047R mutation may be a potential biomarker of sensitivity to everolimus.