ErbB signaling and cell cycle pathways associated with trastuzumab deruxtecan resistance in HER2-positive metastatic breast cancer: a case report

Introduction

Human epidermal growth factor receptor 2 (HER2) overexpression occurs in 15% to 20% of breast cancers (1). Trastuzumab deruxtecan (T-DXd) is the preferred standard second-line therapy for HER2-positive metastatic breast cancer (MBC) (2,3) and demonstrated durable antitumor activity in patients resistant to multiple lines of HER2-targeted therapy (4,5). Despite the remarkable clinical responses observed, the emergence of therapeutic resistance is inevitable, and the underlying resistance mechanisms to T-DXd remain elusive. Here, we present a compelling case of a luminal B, HER2-positive MBC patient who exhibited a dramatic response to T-DXd after five lines of prior HER2-targeted therapy but eventually developed acquired resistance 14 months on therapy. We further report our endeavors to overcome T-DXd resistance assisted by organoid drug screening and the consequential discoveries obtained through next-generation sequencing (NGS) analysis conducted before and after disease progression. These findings provide valuable insights into the potential mechanisms contributing to resistance and aid in guiding treatment selection following the progression on T-DXd. We present this case in accordance with the CARE reporting checklist (available at https://acr.amegroups.com/article/view/10.21037/acr-2025-118/rc).

Case presentation

The patient was a 48-year-old female who presented with a large right breast mass (8.0 cm × 10.0 cm) and widespread metastases involving multiple lymph nodes, liver, bone, and pleura (Figure 1), with no medical history or co-morbidities. There was no history of breast cancer in her family. A breast biopsy confirmed the presence of estrogen receptor (ER)/progesterone receptor (PR)-positive, HER2-positive mixed invasive micropapillary-ductal breast carcinoma. The patient was initiated on first-line therapy with trastuzumab, carboplatin, and paclitaxel and exhibited a partial response, which enabled a modified radical mastectomy to be performed to improve her quality of life. She was transitioned to maintenance therapy with trastuzumab and capecitabine and remained progression-free for 12 months. Following the development of multiple chest wall nodules, second-line therapy with trastuzumab and vinorelbine was initiated, which only sustained a stable disease for 5 months before new growing liver nodules and progressive bone metastases were detected. A third-line regimen comprising trastuzumab, pertuzumab, and nab-paclitaxel was administered, resulting in a partial response and a remarkable progression-free survival exceeding 32 months. The patient subsequently received pyrotinib, palbociclib, and exemestane following the progression of bone metastases. However, the patient experienced grade 3 nausea and vomiting resulting in treatment discontinuation. She was switched to trastuzumab emtansine but exhibited a rapid disease progression with an enlarging gluteal mass within 2 months. Biopsy results identified the presence of ER/PR-negative, HER2-positive metastatic adenocarcinoma of breast origin.

Figure 1 Treatment timeline and clinical course. ER, estrogen receptor; FISH, fluorescence in situ hybridization; HER2, human epidermal growth factor receptor 2; N&V, nausea and vomiting; PFS, progression-free survival; PR, progesterone receptor; RC48, disitamab vedotin; T-DM, trastuzumab emtansine; T-DXd, trastuzumab deruxtecan.

T-DXd was administered as the sixth-line regimen, resulting in a dramatic clinical response with near-complete resolution of the gluteal mass. After 14 months of therapy, the patient developed progressive cervical lymphadenopathy. The pathologic examination reported ER/PR-positive, HER2-positive metastatic carcinoma of breast origin (Figure 2). Patient-derived organoids were generated, and subsequent drug screening was performed, which identified anlotinib plus T-DXd as a potential candidate regimen (Figure 3). She was then treated with anlotinib and T-DXd and had disease control for 6 months before progressive liver metastases were observed. The combination of tucatinib, inetetamab, and eribulin was administered, but the patient experienced further progression in the liver metastases within 2 months. A liver biopsy was performed confirming the presence of metastases. One course of disitamab vedotin (RC48), tislelizumab, and pertuzumab was administered but did not yield any noticeable effect. Another attempt was made with the combination of tucatinib and T-DXd. The combination of fulvestrant, everolimus, pyrotinib, and trastuzumab was administered. However, the patient experienced grade 3 nausea, vomiting and stomatitis resulting in treatment discontinuation. Despite these efforts, the progressive liver metastases persisted the patient was transitioned to hospice care due to worsening symptoms of bone pain, bloating, diarrhea, nausea, and vomiting. She eventually succumbed to the disease 12 months after the emergence of T-DXd resistance, with an overall survival extending to 85 months since the initial diagnosis.

Figure 2 Representative H&E and immunohistochemical staining. (A) Primary breast tumor. (B) Gluteal mass biopsy specimen obtained before the initiation of T-DXd. (C) Cervical lymph node surgically resected specimen obtained after the emergence of T-DXd resistance. Scale bar, 20 µm. ER, estrogen receptor; H&E, hematoxylin and eosin; HER2, human epidermal growth factor receptor 2; PR, progesterone receptor; T-DXd, trastuzumab deruxtecan.

Figure 3 EdU proliferation assays of drug-treated organoids. (A) Fluorescent presentation of cellular structures of the experimental groups; red fluorescence indicates the presence of actively proliferating cells; pink fluorescence indicates the merged blue and red images. Scale bar, 25 µm. (B) The percentages of EdU-labeled proliferative cells in the organoids of the experimental groups. EdU, 5-ethynyl-2'-deoxyuridine; T-DXd, trastuzumab deruxtecan.

Genetic analysis

NGS of 550 cancer-associated genes was performed prior to T-DXd administration (HiSeq X) and revealed the presence of PIK3CA^E545K mutation (VAF, 35.9%), ATM^P2699S mutation (VAF, 79.35%), TOP2A^I317V alteration (VAF, 15.8%), ERBB2 amplification (15.5×), and CDK12 amplification (12.5×). After the emergence of T-DXd resistance, NGS analysis was repeated (HiSeq X) and identified new alterations, including ARAF^A110T alteration (VAF, 28.13%), along with amplifications in MYC (2.88×), MDM2 (3.5×), PRKDC (2.88×), TYMS (3.25×), and YES1 (4.0×). NGS was again repeated following resistance to T-DXd plus anlotinib (HiSeq X) and found new amplification in KRAS (10.16×), FLT1/3 (4.55×), CD274 (3.7×), and PDCD1LG2 (3.1×), as well as increased amplifications in MYC (8.29×), TYMS (5.4×), and YES1 (4.72×). Tumor mutation burden increased from 2.593 to 6.000 Muts/Mb. The KEGG pathway analysis comparing genetic alterations identified prior to and after T-DXd resistance revealed enrichment of pathways implicated in the ErbB signaling (P<0.002) and cell cycle (P<0.006) (Figure 4).

Figure 4 Genetic alterations associated with resistance to T-DXd. (A) Venn diagram of genetic alterations detected before and after T-DXd resistance. (B) Genetic alterations were detected across the three targeted gene sequencings. Each column represents a sample, and each row a gene. Colored blocks indicate gene mutations; grey blocks represent wild-type. The top bar plot shows the number of mutated genes per sample (0–3 scale). Right-side percentages indicate the mutation frequency of each gene across samples. (C) KEGG enrichment results of newly emerged mutated genes identified after treatment resistance, compared to those before treatment. KEGG, Kyoto Encyclopedia of Genes and Genomes; T-DXd, trastuzumab deruxtecan.

Data collection and organoid generation, drug treatments, and sensitivity analysis are seen in Appendix 1.

All procedures performed in this study were in accordance with the Declaration of Helsinki and its subsequent amendments. Tumor samples were collected with ethical approval from the Ethics Committee of Guangdong Provincial People’s Hospital, Southern Medical University (accreditation No. KYQ202102903). Written consent was obtained from the patient’s next of kin to have the anonymized information published in this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Discussion

Despite encouraging breakthroughs in the management of HER2-positive MBC, the development of resistance to HER2-targeted therapy is inevitable. At present, research on the mechanisms of resistance to T-DXd or the drug of choice for patients resistant to T-DXd is scarce. The DAISY trial was the first to investigate the mechanisms underlying T-DXd resistance by comparing matched samples acquired at baseline and the onset of disease progression in MBC patients. The study unveiled a strong correlation between the level of HER2 expression and the antitumor efficacy of T-DXd, with 65% of patients resistant to T-DXd displaying a reduction in HER2 expression. Loss of function mutations in SLX4 may also contribute to secondary resistance against T-DXd (6). Zou et al. revealed the molecular mechanisms underlying T-DXd resistance in HER2-low breast cancer, and provided a new strategy to overcome T-DXd resistance by inhibiting the interaction between crVDAC3 and HSPB1 protein (7). In addition, a paired genomic analysis conducted on HER2-mutated, non-small-cell lung cancer cases suggested the functional loss of Rb1 could potentially confer acquired resistance to T-DXd (8).

Corresponding with the findings of the DAISY trial, our patient demonstrated a decrease in HER2 expression from an immunohistochemistry score of 3+ to 2+. As shown in Figure 4A,4B, the mutation profiles across the three samples were compared in terms of similarity and individual mutational characteristics. A clear difference was observed in the mutational landscape before and after T-DXd treatment, with only two shared mutated genes, while the post-treatment samples (post T-DXd_1 and post T-DXd_2) harbored 10 and 16 unique mutations, respectively. These findings reflect tumor evolution following the development of drug resistance. Pathway analysis further revealed enrichment of pathways implicated in the ErbB signaling (KRAS, ARAF, MYC) and cell cycle (MDM2, MYC, PRKDC) might be associated with T-DXd resistance (Figure 4). Anomalous signaling within the ErbB pathway has been involved in the initiation and progression of breast cancer (9). The persistence amplification of KRAS and MYC, as well as the mutation of ARAF, observed following progression on T-DXd, may suggest a dysregulated activation of the Ras/MEK/ERK pathway independent of ErbB3 and ErbB2 phosphorylation (10). Prior research has shown that Ras/MEK/ERK pathway activation can decrease PTEN expression, thereby engendering carcinogenesis and bestowing resistance to chemotherapeutic interventions (11,12). Aberrant signaling within the cell cycle pathway, exemplified by MDM2 amplification, may also contribute to resistance against T-DXd. MDM2 serves as a key negative regulator of p53 and facilitates the G1-S transition of the cell cycle (13). Overexpression of MDM2 has been observed to induce the degradation of the Rb protein (14), aligning with earlier reports associating Rb1 loss with secondary resistance to T-DXd (8).

To explore potential strategies for overcoming T-DXd resistance, in vitro drug screening employing patient-derived organoids was performed. Various targeted agents were assessed, including tyrosine kinase inhibitors (anlotinib, pyrotinib), a CDK4/6 inhibitor (palbociclib), and a PD-1 inhibitor (pembrolizumab). The combination of anlotinib and T-DXd exhibited the highest rate of cell mortality and achieved a disease control period lasting 5 months. Anlotinib exerts its effects by inhibiting tumor angiogenesis and proliferation and has demonstrated notable antitumor activity in HER2-negative MBC (15,16), representing a potential option to overcome T-DXd resistance by targeting different tumorigenesis pathways such as the VEGF signaling pathway. Additionally, targeting the Ras/MEK/ERK pathway (17) and cell cycle proteins (18) could serve as alternative approaches to address T-DXd resistance, particularly in MYC-driven cancer cells, which have shown heightened sensitivity to CDK7 or CDK9 inhibition (19). The limited efficacy observed with HER2-targeted tyrosine kinase inhibitors may be attributed to the activation of downstream pathways within the ErbB signaling cascade. The absence of antitumor efficacy of CDK4/6 inhibitors in T-DXd-resistant organoids could be explained by the loss of Rb and PTEN, which potentially confers resistance to CDK4/6 inhibitors (20). Despite preclinical evidence showing potentiated antitumor immunity in HER2-amplified colorectal cancer cells treated with T-DXd (21), our observations did not endorse the concurrent use of immunotherapy and T-DXd. Ongoing clinical trials investigating the combination of immune checkpoint inhibitors with T-DXd in HER2-positive MBC have yet to report results. A decrease in PD-L1 expression was detected in HER2-amplified MBC following treatment with T-DXd in the DAISY trial (6). Activation of the Ras/MEK/ERK pathway was associated with PTEN loss, resulting in subsequent impaired tumor immune responses (12,22). The organoid drug screening of PD-1 inhibitor plus T-DXd revealed no notable antitumor activity, further corroborated by the lack of patient response to the combination of PD-1 inhibitor and RC48.

Conclusions

In summary, our report highlights that T-DXd resistance may be attributed to dysregulation in the ErbB signaling and cell cycle pathways. In vitro experiments are being conducted to ascertain the potential benefits of targeted inhibition of angiogenesis or cell cycle proteins in overcoming T-DXd resistance.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://acr.amegroups.com/article/view/10.21037/acr-2025-118/rc

Peer Review File: Available at https://acr.amegroups.com/article/view/10.21037/acr-2025-118/prf

Funding: This work was supported by the National Natural Science Foundation of China (No. 82303848), the Medical Scientific Research Foundation of Guangdong Province (No. A2023020), the Scientific Research Project of Guangdong Provincial Bureau of Traditional Chinese Medicine (No. 20241003), the Xisike-Hengrui Cancer Research Fund (No. Y-HR2022QN-0354), and the Lianyungang Yixing Medical Health Foundation (No. YX-XM2022A15).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://acr.amegroups.com/article/view/10.21037/acr-2025-118/coif). X.Z.L. was employed by the company Burning Rock Biotech Ltd. at the time of writing this report. H.L. is an employee of Biomedical Laboratory, Jingke BioTech Group, Guangzhou, China. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the Declaration of Helsinki and its subsequent amendments. Tumor samples were collected with ethical approval from the Ethics Committee of Guangdong Provincial People’s Hospital, Southern Medical University (accreditation No. KYQ202102903). Written consent was obtained from the patient’s next of kin to have the anonymized information published in this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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