DNA Damage and Nuclear Anaplasia Induced by Trastuzumab Deruxtecan in Cancer Cells with Variable HER2 Expression and Homologous Recombination Deficiency Status

So Hyeon Kim; Yoonjung Park; Ahrum Min; Hye Yeon Park; Yu-Jin Kim; Sujin Ham; Jiwon Koh; Seongyeong Kim; Dae-Won Lee; Han Suk Ryu; Jin-Soo Kim; Kyung-Hun Lee; Seock-Ah Im

doi:10.4143/crt.2025.201

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Original Article General DNA Damage and Nuclear Anaplasia Induced by Trastuzumab Deruxtecan in Cancer Cells with Variable HER2 Expression and Homologous Recombination Deficiency Status: So Hyeon Kim¹, Yoonjung Park¹, Ahrum Min¹, Hye Yeon Park², Yu-Jin Kim¹, Sujin Ham¹, Jiwon Koh^1,3, Seongyeong Kim¹, Dae-Won Lee^1,2,4,5, Han Suk Ryu^1,3, Jin-Soo Kim⁶, Kyung-Hun Lee^1,4,5, Seock-Ah Im^1,2,4,5; Cancer Research and Treatment : Official Journal of Korean Cancer Association 2026;58(2):407-422.
DOI: https://doi.org/10.4143/crt.2025.201
Published online: August 4, 2025

¹Cancer Research Institute, Seoul National University, Seoul, Korea

²Department of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea

³Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea

⁴Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea

⁵Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea

⁶Department of Internal Medicine, Seoul Metropolitan Government-Seoul National University Boramae Medical Center, Seoul, Korea

Correspondence: Seock-Ah Im, Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, 101, Daehak-ro, Jongno-gu, Seoul 03080, Korea
Tel: 82-2-2072-0850 E-mail: moisa@snu.ac.kr

^*So Hyeon Kim and Yoonjung Park contributed equally to this work.

• Received: February 21, 2025 • Accepted: June 2, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract

Purpose
Human epidermal growth factor receptor 2 (HER2) is amplified or overexpressed in various malignancies, including breast and gastric cancers, and is associated with poor prognosis. Although HER2-targeted therapies, such as trastuzumab, improve outcomes in HER2-positive tumors, resistance often develops, and HER2-low tumors remain largely untargeted. Trastuzumab deruxtecan (T-DXd; DS-8201a) is a HER2-targeted antibody-drug conjugate with potent activity in HER2-positive and HER2-low tumors. This study evaluates its antitumor mechanisms and efficacy in HER2-positive, HER2-low, and homologous recombination deficiency (HRD)–associated models.
Materials and Methods
Effects of T-DXd were assessed in cancer cell lines with diverse HER2 expression and HRD status. In vivo efficacy was evaluated using a xenograft model derived from HER2-low SNU-601 gastric cancer cells.
Results
T-DXd reduced HER2 phosphorylation and downstream signaling (AKT, ERK) in HER2-positive cells. It induced DNA damage accumulation, as evidenced by increased γH2AX and p-Chk1 expression, and triggered apoptosis through cleaved poly(ADP-ribose) polymerase and caspase-3 activation, confirmed by annexin V staining. Similar effects were observed in HER2-low cells, with greater sensitivity in HRD cells. In xenografts, T-DXd reduced tumor volume by up to 80% at 4 mg/kg and 10 mg/kg. Histological analyses showed decreased Ki-67 and increased apoptosis. Furthermore, T-DXd induced G2/M cell cycle arrest and nuclear anaplasia, suggesting disruption of chromosomal stability as a potential antitumor mechanism. No significant toxicity, including body weight loss, was observed.
Conclusion
These findings highlight T-DXd’s effectiveness in HER2-low and HRD tumors, supporting its broader clinical application, including strategies targeting DNA damage repair pathways.
Key words: Trastuzumab deruxtecan, DNA damage, Nuclear anaplasia, Homologous recombination deficiency, HER2

Introduction

Human epidermal growth factor receptor 2 (HER2), also known as ErbB2, belongs to the epidermal growth factor receptor (EGFR) family tyrosine kinases and promotes cell proliferation, survival, and angiogenesis. Amplification or overexpression of HER2 is observed in various cancers, such as gastric, colorectal, bladder, ovarian, biliary, and breast cancers. Especially, HER2 amplified or overexpressed cancers account for 20%-25% of breast and gastric cancer patients and are associated with advanced stage [1]. Therefore, HER2 is considered a crucial therapeutic target.

HER2-targeted drugs have been developed and prolonged the progression-free survival (PFS) and overall survival (OS) in HER2-positive cancer patients. For example, trastuzumab, an anti-HER2 monoclonal antibody, has been used with chemotherapy as the standard care for HER2-positive metastatic breast and gastric cancers. Lapatinib, a small molecule inhibitor of EGFR and HER2, reduced the disease progression in combination with capecitabine for the HER2-positive metastatic breast cancer [2]. Although these HER2-targeted drugs improve clinical outcomes in patients with HER2-positive cancer, unfortunately, some patients do not respond to these drugs. Even in responders, the disease progresses due to acquired resistance. Thus, the development of novel HER2-targeted therapy to expand its application into various HER2-positive cancer patients is needed.

Trastuzumab deruxtecan (T-DXd; DS-8201a), is a novel HER2-targeted antibody-drug conjugate (ADC). It is an anti-HER2 human monoclonal antibody generated for the same amino acid sequence as trastuzumab, conjugated with DX-8951f derivative (DXd), a topoisomerase 1 (TOP1) inhibitor by a tetrapeptide linker. The linker of T-DXd is stable in plasma, but after internalization into the cells, it is cleaved by cathepsins, which is a lysosomal enzyme overexpressed in cancer cells [3]. In phase 2 clinical trials, T-DXd significantly prolonged PFS in heavily pretreated patients with HER2-positive metastatic gastric and breast cancers, as well as various HER2-expressing solid tumors [4-7]. Moreover, in phase 3 trials, it improved PFS and OS in HER2-positive metastatic breast cancer patients [8,9]. Furthermore, it prolonged PFS and OS compared with chemotherapy in heavily pretreated patients with HER2-low breast cancer, who have not been previously considered as optimal candidates for HER2-targeted drugs [8,10].

Several preclinical studies reported that T-DXd is effective even in HER2-low cancer cells since the average drug-to-antibody ratio of T-DXd is higher than other HER2-ADCs and has a potent cytotoxic bystander effect [3]. The antitumor effect of T-DXd is caused by the payload, a TOP1 inhibitor. Recent studies suggested that TOP1 inhibitor is effective in the treatment of tumors with homologous recombination deficiency (HRD) [11]. However, the mechanism of action of T-DXd and its association with HRD status are still unclear. Thus, we evaluated the antitumor effect of T-DXd in human cancer cell lines with a wide range of HER2 expression and HRD status, and explored the mechanism of action.

Materials and Methods

1. Reagents and antibodies

T-DXd (DS-8201a) was kindly provided by Daiichi Sankyo Co., Ltd. through the research proposal. The drug stock was diluted with ABS (10 mM acetate buffer, 5% sorbitol, pH 5.5) and stored at –80℃.

Antibodies against TDP1 (ab4166) and TOP1 (ab109374) were purchased from Abcam. Antibody against poly(ADP-ribose) polymerase (PARP; 556494) was purchased from BD Biosciences and phospho-RPA32 (A300-245A) was purchased from Bethyl Laboratories. Antibodies against AKT (#9272), Caspase-3 (#9662), HER2 (#2242), p44/42 mitogen-activated protein kinase (MAPK) (#9102), phospho-AKT (S473) (#9271), phospho-Chk1 (S345) (#2348), phospho-HER2 (Y1221/1222) (#2249), phospho-histone H2A.X (#9718), and phospho-p42/44 MAPK (T202/Y204) (#9216) were purchased from Cell Signaling Technology. Antibodies against actin (A3853) were purchased from Sigma-Aldrich and cyclin B1 (sc-752) was purchased from Santa Cruz Biotechnology. Secondary antibodies Alexa Fluor conjugate (A-11001 and A-11012) were purchased from Thermo Fisher Scientific Inc.

2. Cell lines and cell cultures

NCI-N87 Cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). SK-BR-3, HCC-1419, ZR-75-1, MDA-MB-231, Capan-1, SNU-601, SNU-638, SNU-668, SNU-216, and SNU-484 were obtained from the Korean Cell Line Bank (Seoul, Korea), which were banked and passaged for less than 3 months before use. All cell lines were maintained in RPMI-1640 medium (LM011-01, Welgene) supplemented with 10% fetal bovine serum and 10 μg/mL gentamicin (15750078, Thermo Fisher Scientific Inc.) in a humidified atmosphere of 5% CO₂ at 37°C.

3. Histopathologic review of breast cancer after T-DXd treatment

In compliance with the approval from the Institutional Review Board (IRB) of Seoul National University Hospital (IRB No. 2310-165-1480), we reviewed the histopathologic features of breast cancer before and after T-DXd treatment. The patient had a histologic grade III, hormone receptor–negative HER2-positive metastatic breast cancer which progressed after 1st line trastuzumab with chemotherapy and 2nd line T-DM1, she received T-DXd treatment after disease progression on prior HER2-targeted therapies, achieving partial response and remaining progression-free for 11 months.

4. Immunohistochemistry

Immunohistochemistry (IHC) was performed using paraffin‑embedded cell pellets obtained from 150 cm² flasks, and xenograft tissue, fixed in 10% neutral buffered formalin. HER2 IHC score was interpreted using membranous staining intensity and percentage of HER2-stained cells according to American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) 2023 guidelines [12]. Cell proliferation was assessed by Ki-67 IHC using anti-rabbit polyclonal antibody against Ki-67 (MA5-14520, Thermo Fisher Scientific Inc.) at a dilution of 1:100. Apoptosis was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay-based ApopTag In Situ Apoptosis Detection Kit (S7101, Sigma-Aldrich). A pathologist (J.K.) interpreted Ki-67 and TUNEL as percentages of positive tumor cells.

5. Colony formation assay

Cells were trypsinized to a single-cell suspension and seeded into 6-well plates at a density of 1-8×10³. Cells were allowed to attach for over 2 days. Then, cells were treated with T-DXd. Two weeks later, colonies were fixed and stained with 50% methanol, 10% glacial acetic acid, and 0.1% Coomassie Brilliant Blue R-250 solution for 2 hours. The number of colonies was normalized with the value of the control dose. IC₅₀ was calculated using SigmaPlot version 10.0 (Systat Software Inc.).

6. Western blot analysis

Cells were collected after T-DXd treatments and incubated in extraction buffer (50 mM Tris-HCl; pH 7.4, 150 mM NaCl, 1% NP40, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mM sodium fluoride, 1 mM sodium pyrophosphate, 2 mM phenylmethylsulfonyl fluoride, 1 mg/mL pepstatin A, 0.2 mM leupeptin, 10 μg/mL aprotinin, 1 mM sodium vanadate, 1 mM nitrophenylphosphate, and 5 mM benzamidine) on ice for 30 minutes. After cell lysis, the isolated protein was quantified using the Bradford method [13]. Subjected to SDS polyacrylamide gel electrophoresis, and then transferred onto a nitrocellulose membrane. After being blocked, the membrane was incubated with primary polyclonal antibodies overnight at 4℃, and subsequently incubated with the secondary antibody for 1 hour at room temperature. The protein bands were detected using an enhanced electrochemiluminescence detection system (ECL system, Amersham Pharmacia Biotech).

7. Genomic DNA extraction and quantitative polymerase chain reaction

Genomic DNA was extracted using the DNeasy purification kit (51304, Qiagen) according to the manufacturer’s instructions. A total of 20 ng of DNA were run in a IQTM5 Optical Module (Bio-Rad). The thermal cycle conditions were as follows: 10-minute denaturation at 95℃, and the next 40 cycles were carried out with 5 minutes at 60℃ followed by 15 seconds at 95℃. All data from all samples were normalized to actin and re-normalized to a reference sample with a neutral copy number. The actin primer was synthesized by Macrogen, ErbB2 primer was synthesized by Bioneer as described in S1 Table. All polymerase chain reaction analyses and copy number calculations were conducted in triplicate.

8. Cell surface fluorescence measurement

To investigate cell surface-bound T-DXd, cells were incubated with 10 μg/mL T-DXd on ice for 1 hour and then washed to remove unbound antibodies. Cells were then incubated in complete media at 37°C for indicated periods of time. T-DXd bound on cell surface was stained with Alexa Fluor 488-labeled antibody against human IgG and analyzed by flow cytometry. Receptor-antibody complex internalization was calculated as percent mean fluorescent intensity (MFI) after subtracting the background value of MFI derived from the untreated control.

9. Immunofluorescence assay

Cells were cultured on poly-L-lysine (P4707, Sigma-Aldrich) coated coverslips in 6-well plates and fixed with 3.7 % formaldehyde. Fixed cells were then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) and then blocked in 2% bovine serum albumin (BSA) in PBS. Primary antibodies were incubated for 2 hours at 37℃ followed by secondary antibody incubation for 1 hour at 37℃. Cells were counterstained with DAPI, mounted on glass slides with mounting medium (S3025, Dako), and allowed to cure overnight. To investigate the morphological characteristics of the nucleus, cells were only stained with DAPI. Image acquisition was performed on a Zeiss LSM 800 laser scanning microscope.

10. Cell cycle analysis

Cells treated with T-DXd were grown to 60% to 70% confluence and trypsinized. Cells were fixed in 80% ethanol and then stored at –20℃ for at least 24 hours. After centrifugation, the pellet was resuspended in PBS containing 0.1% BSA and 10 μg/mL RNase A (12091-021, Invitrogen) at 37℃ for 30 minutes. Next, cells were treated with 20 μg/mL propidium iodide (P4864, Sigma-Aldrich). DNA content and cell cycle analysis of at least 10,000 cells per experimental group were done on a fluorescence-activated cell sorting (FACS) Calibur flow cytometer (BD Biosciences).

11. Annexin V assay

To quantify apoptotic cells, annexin V assay was performed using annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (556547, BD Pharmingen) according to the manufacturer’s instructions. Cells were collected after T-DXd treatments, and resuspended in binding buffer, followed by staining with annexin V–FITC and PI solution at 37℃ in the dark. Analyses were then performed using a FACS Calibur flow cytometer (BD Biosciences).

12. Alkaline comet assay

Alkaline comet assay was performed using the CometAssay kit (4250-050-K, Trevigen) following the manufacturer’s instructions. Briefly, cell suspension was carefully mixed with molten LMAgarose. After solidification, slides were immersed in Lysis Solution for 40 minutes, at 4℃, and Alkaline Unwinding Solution for 30 minutes at room temperature in the dark. Electrophoresis was performed in alkaline electrophoresis buffer for 30 minutes. Slides were treated with 70% ethanol for 5 minutes at room temperature. After air drying, cells were stained with SYBR Green, and coverslips were then mounted onto slides using a mounting medium (Dako). Comet images were captured using a Zeiss LSM 800 laser scanning microscope.

13. Topoisomerase 1 assay on DNA

Cells were treated with or without T-DXd, then nuclear proteins were isolated using a Subcellular Protein Fractionation Kit (78840, Thermo Fisher Scientific Inc.) following the manufacturer’s instructions. TOP 1 activity was determined in isolated nuclear proteins by using a Topoisomerase I Assay Kit (TG1015-1, TopoGEN Inc.) according to manufacturer’s protocols. Briefly, nuclear protein extract was incubated with supercoiled DNA in 1× recombinant topo 1 reaction buffer for 60 minutes at 37℃ and terminated by adding stop loading buffer. Samples were then loaded on a 0.8% agarose gel and run in 1× TAE buffer. Following staining with ethidium bromide for 15 minutes and destaining in distilled water, gels were photographed using a UV transilluminator Gel Doc XR+ System (Bio-Rad).

14. In vivo activity in a xenograft model

All animal experiments were conducted in the animal facility of Seoul National University under institutional guidelines and prior approval from the Institutional Animal Care and Use Committee (IACUC No.: 18-0252-S1A0). SNU-601 cells (5×10⁷ per mouse) were subcutaneously injected into the right flanks of female BALB/c nude mice (Orient-Bio Inc.). Once tumors had reached 200 mm³, mice were randomly assigned to treatment and control groups. On day 0, mice received an intravenous injection of vehicle or T-DXd (0, 4, 10 mg/kg in 100 μL vehicle). We used seven mice for control, seven for 4 mg/kg dosage, and 9 for 10 mg/kg dosage. Tumor volumes and mouse body weights were measured three times per week. Mice were sacrificed with CO₂ if tumors became necrotic or grew to a volume of 1,000 mm³. Tumor volume was defined as 1/2×length×width².

15. Statistical analysis

Categorical variables were summarized as frequencies and percentages, whereas continuous variables were presented as medians with ranges. Group differences in categorical variables were assessed using the chi-square test or Fisher’s exact test, as appropriate. For continuous variables, the t test, Mann-Whitney U test, or the Kruskal-Wallis test was used as appropriate. A p-value < 0.05 was considered statistically significant.

In vivo tumor volume and body weight measurements were collected at multiple time points and are shown as mean±standard error. Between-group comparisons of tumor growth curves were analyzed by two-way repeated-measures ANOVA. A p-value < 0.05 was considered statistically significant.

Results

1. T-DXd inhibits cell proliferation through induction of apoptosis in human cancer cell lines expressing wide range of HER2

To determine the correlation between HER2 expression and antitumor effect of T-DXd, we confirmed HER2 status, in terms of HER2 gene amplification and protein expression of HER2, in various human cancer cell lines (S2 Fig.). We grouped the cell lines into three categories according to the HER2 status. HER2-positive cell lines (SK-BR-3, HCC1419, NCI-N87, and SNU-216) were defined as cell lines with IHC 3+, IHC 2+ with HER2 gene amplification according to ASCO/CAP 2023 guidelines or high protein expression of HER2 [12]. Cell lines with neutral HER2 copy, IHC score 2+, 1+ , and low protein expression were classified as HER2-low group (ZR-75-1, SNU-484, SNU-601, SNU-638, SNU-668, SNU-4257 and Capan-1). A cell line without detectable HER2 expression was classified as HER2-negative (MDA-MB-231) (Table 1).

Next, we evaluated the antitumor effect of T-DXd using a colony formation assay. HER2-positive breast cancer cell lines (SK-BR-3 and HCC1419) exhibited high sensitivity with IC₅₀ values of 1.94 ng/mL and 18.3 ng/mL, respectively. The HER2-low cell line (ZR-75-1) showed moderate sensitivity with an IC₅₀ of 30.25 ng/mL. Notably, the cytotoxic effect of T-DXd was not observed up to 100 ng/mL in the HER2-negative cell line (MDA-MB-231) (Table 1, Fig. 1A).

Compared with breast cancer cell lines, gastric and pancreatic cancer cell lines exhibited a relatively lower sensitivity to T-DXd. HER2-positive gastric cancer cell lines (NCI-N87 and SNU-216) had IC₅₀ values of 15.3 ng/mL and 102 ng/mL, respectively. HER2-low gastric and pancreatic cancer cell lines had IC₅₀ values ranging from 143.8 ng/mL to over 500 ng/mL. Capan-1 and SNU-484, which had relatively moderate HER2 expression, showed intermediate IC₅₀ values of 143.8 ng/mL and 219.8 ng/mL, respectively (Table 1, Fig. 1B). Interestingly, Capan-1, harboring a BRCA2 mutation, exhibited higher sensitivity compared to SNU-484, which has no mutations in HRR (homologous recombination repair)–related genes. Additionally, SNU-601, which harbors RAD51 promoter methylation [14], exhibited sensitivity comparable to SNU-484 (IC₅₀ values of SNU-601, 244.7 ng/mL) despite its lower HER2 expression (Table 1, Fig. 1B). These results suggest that the cytotoxicity of T-DXd was associated with HRR status.

After treatment of T-DXd, annexin V–positive population was significantly increased (Fig. 1C and D). Furthermore, T-DXd induced cleavage of PARP and caspase-3 in HER2-positive breast cancer cell lines (Fig. 1E) and HER2-positive gastric cancer cell lines (Fig. 1F). Notably, T-DXd also increased the annexin V–positive population and induced cleavage of PARP and caspase-3 in some HER2-low cell lines, including ZR-75-1, SNU-484, SNU-601, and Capan-1 (Fig. 1C and D, S3 Fig.). While, in cell lines with IC₅₀ values > 100 or 500 ng/mL, the annexin V–positive population and cleavage of PARP and caspase-3 were not changed (Table 1, Fig. 1). Taken together, these results indicate that T-DXd causes apoptosis and exhibits an anti-proliferative effect in not only HER2-positive cell lines but also HER2-low cell lines.

2. Internalization of T-DXd occurs regardless of HER2 expression, and T-DXd suppresses HER2 signal transduction

ADC is internalized into the cells after binding to the target receptor, and the efficacy of ADC is influenced by the amount of drug internalized and payload released into the cells [3]. Therefore, we inferred the internalization of T-DXd by measuring the amount of T-DXd attached to the cell surface over time. In HER2-positive cell lines, the level of cell surface binding T-DXd was higher than in other cell lines, and the level of cell surface binding T-DXd was lowest in HER2-negative cell line (Fig. 2A). And, except in HER2-negative cell line, internalization of T-DXd was increased over time in HER2-positive and HER2-low cell lines (Fig. 2B). This data demonstrated that T-DXd internalized into the cell regardless expression level of HER2.

Trastuzumab binds to HER2 extracellular domain and inhibits HER2-mediated signal transduction [15]. To determine whether the HER2 signaling inhibition by T-DXd is related to the HER2 expression levels, we examined the expression of HER2 downstream molecules followed by T-DXd treatment in human cancer cell lines with variable HER2 expression. T-DXd suppressed phosphorylation of AKT and ERK in HER2-positive breast cancer cell lines. And phosphorylation of HER2 was decreased after T-DXd treatment in HCC1419. In contrast, we did not observe these changes in HER2-low and HER2-negative breast cancer cell lines (Fig. 2C). Consistent with observations in HER2-positive breast cancer cell lines, phosphorylation of HER2, AKT, and ERK were decreased after T-DXd treatment in HER2-positive gastric cancer cell lines (Fig. 2D, S4 Fig.). Collectively, we demonstrated that T-DXd was internalized into cancer cells regardless of HER2 expression although there is a difference in the extent, but suppressed HER2 signaling only in HER2-positive cells.

3. T-DXd causes DNA damage accumulation

TOP1 inhibitor induces cell death through the accumulation of DNA damage [16]. Therefore, we hypothesized that T-DXd induces cell death via DNA damage accumulation by TOP1 inhibitor payload. To confirm this hypothesis, we verified the expression of DNA damage markers. T-DXd increased p-RPA, p-Chk1, and γH2AX expression in HER2-positive breast and gastric cancer cell lines (Fig. 3). And γH2AX expression was increased after T-DXd treatment in HER2-low breast (Fig. 3A), pancreatic, and some of gastric cancer cell lines (SNU-484 and SNU-601) (Fig. 3B, S5 Fig.). Furthermore, the results of the comet assay showed that tail moment, which represents DNA fragmentation, was significantly increased in these cell lines (Fig. 3C and D, S5 Fig.).

Notably, we observed that high level of tail moment in the HRR-gene deficient HER2-low cell lines (Capan-1 and SNU-601) compared to the HER2-low cell line with wild-type HRR genes (SNU-484). Furthermore, RAD51 and γH2AX co-localized in SK-BR-3, HCC1419, ZR-75-1, NCI-N87 and SNU-216, but not in SNU-601 cell lines (Fig. 3E and F).

These results indicate that T-DXd induces DNA damage and that HRD status is one of the important factors influencing its antitumor effect.

4. T-DXd induces cell cycle arrest in G2/M phase and nuclear anaplasia

We investigated the influence of DNA damage to cell cycle progression. In response to T-DXd, the percentage of cells in the G2/M phase increased in HER2-positive and HER2-low breast cancer cell lines (Fig. 4A). Moreover, T-DXd increased proportion of cells in the G2/M phase in HER2-positive gastric and some of HER2-low gastric and pancreatic cancer cell lines (SNU-484 and Capan-1) (Fig. 4B). Treatment with T-DXd did not significantly increase the proportion of SNU-601 cells in the G2/M phase of the cell cycle within 120 hours. However, a significant increase in the G2/M cell population was observed after 10 days of T-DXd treatment (Fig. 4B, S6 Fig.). Consistent with the cytotoxicity of T-DXd, breast cancer cell lines exhibited a higher percentage of G2/M phase cell population than gastric cancer cell lines, even though it was treated with a lower concentration of the T-DXd (Fig. 4A and B). To confirm this data, we confirmed the expression of the regulator of the G2/M cell cycle. T-DXd increased cyclin B1 and p-CDK1 in HER2-positive and HER2-low breast cancer cell lines (Fig. 4C). As observed in breast cancer cell lines, cyclin B1 and p-CDK1 increased after T-DXd treatment, in HER2-positive and some of HER2-low gastric (SNU-484) and pancreatic (Capan-1) cancer cell lines (Fig. 4D, S6 Fig.).

If DNA-damaged cells enter mitosis and divide even before DNA damage is completely repaired, it can lead to aberrant chromosome [14]. Since T-DXd caused DNA damage accumulation (Fig. 3), we investigated the effect of T-DXd on chromosomal stability. After T-DXd treatment, in HER2-positive breast (Fig. 4E) and gastric (Fig. 4F) cancer cell lines, we observed marked nuclear enlargement and abnormal nuclear conformation, which were suggestive of nuclear anaplasia. Furthermore, T-DXd induced abnormal nuclear conformation in HER2-low breast (Fig. 4E) and gastric cancer cell lines (Fig. 4F, S7 Fig.). Interestingly, we also observed a similar morphologic tendency in a patient with HER2-positive breast cancer; compared to the pre-T-DXd breast cancer tissue, there were more profound nuclear anaplastic features in the tumor cells after the T-DXd treatment as shown in Fig. 4G. Moreover, aneuploidy was increased in HER2-positive breast cell lines and NCI-N87, HER2-positive gastric cancer cell line, and ZR-75-1, HER2-low expressed cell line after T-DXd treatment (S8 Fig.). Taken together, these results suggest that DNA damage accumulation induced by T-DXd may cause aberrant chromosomal segregation, which is represented by marked nuclear anaplastic features.

5. T-DXd suppresses TDP1 level and TOP1 activity

TOP1 and TDP1 are associated with the formation of TOP1 cleavage complex (TOP1cc), which is caused by TOP1 inhibitor [16]. Thus, we confirmed the expression of the TOP1 and TDP1. TDP1 expression was decreased after T-DXd treatment in HER2-positive breast cancer cell lines, but there was no change in the TOP1 expression (S9 Fig.). Since there was no correlation between the TDP1 level and the T-DXd sensitivity, we inferred the TOP1 activity by evaluating the conversion of supercoiled DNA into relaxed DNA. In HER2-positive cell lines, relaxed DNA was significantly decreased (S9 Fig.). This data indicates that T-DXd suppresses the TOP1 activity.

6. T-DXd inhibits tumor growth in a xenograft model

To test whether T-DXd can suppress tumor growth even in HER2-low expressing cells in vivo, HER2-low SNU-601 gastric cancer cells were subcutaneously injected into BALB/c nude mice, when tumor volume had reached 200 mm³, the mice received a single dose of either the vehicle or the indicated dose of T-DXd. T-DXd successfully inhibited tumor growth even at a low dose (4 mg/kg) without body weight loss (Fig. 5A and B). Furthermore, T-DXd reduced the expression of Ki-67 and increased TUNEL (Fig. 5C) with statistical significance (Fig. 5D). These results indicated that T-DXd inhibits tumor growth and induces apoptotic cell death in HER2-low xenograft model in vivo.

Discussion

HER2-targeted drugs, such as trastuzumab, lapatinib, and T-DM1, significantly improved the clinical outcome in HER2-positive breast and gastric cancer patients, but not in patients with HER2-low tumors [3,5,17]. Unlike these drugs, T-DXd, a novel HER2-targeted ADC, showed considerable efficacy in patients with a wide range of HER2-expressing solid tumors and support the potential role of T-DXd as a tumor-agnostic therapy for patients with HER2-expressing solid tumors [6]. In previously treated HER2-positive breast cancer patients, the median duration of PFS (16.4 months) was longer than in previously treated HER2-positive gastric cancer patients (5.6 months) [4,5]. Concordance with clinical data, our data showed that T-DXd inhibits cell proliferation and induces the cell death in HER2-low cell lines as well as HER2-positive cell lines. Additionally, the IC₅₀ values in breast cancer cell lines were lower than those in gastric cancer cell lines. Expectedly, level of T-DXd binding on cell surface was higher in HER2-positive cell lines than in HER2-low cell lines and HER2-negative cell line. Interestingly, T-DXd was internalized over time in both HER2-positive cell lines and HER2-low cell lines. Collectively, these results indicate that T-DXd can be internalized effectively even in cells expressing low levels of HER2, and can potentially exert antitumor efficacy irrespective of HER2 expression level.

In general, the individual components of the ADC, namely the antibody and cytotoxic agent, are considered as factors determining the sensitivity of ADC [18]. Thus, we examined the mechanism of action for each component of the T-DXd. We observed that T-DXd strongly inhibits HER2 signal cascade in HER2-positive breast cell lines, as well as in HER2-positive gastric cancer cell lines which are addicted to the HER2 signal pathway. In addition, T-DXd also showed antitumor effect in HER2-low cancer cells without significant changes in HER2 downstream signal transduction molecules. This result indicates that although monoclonal antibody component of T-DXd contributes to the antitumor effect, there may exist other action mechanisms of T-DXd. Our findings indicate that T-DXd exerts potent inhibitory effects of cancer cell growth regardless of HER2 expression, largely due to its TOP1 inhibitor payload. This phenomenon is especially pronounced in HRD tumor cells, which are inherently more susceptible to TOP1-mediated DNA damage.

Some of ADCs, such as immunoliposomes, gene-directed enzyme prodrug therapy, and radioimmunoconjugates, can kill tumor cells with low expression of antigens which are located proximal to tumor cells with high expression. This phenomenon is termed the “bystander killing effect” [19]. T-DXd showed bystander killing effect in HER2-negative cells neighboring HER2-positive cells since the payload of T-DXd has high membrane-permeability. This result indicates that the antitumor effect of T-DXd might rely on the payload of T-DXd, which is a TOP1 inhibitor. TOP1 inhibitors induce DNA damage through the accumulation of TOP1cc which functions as an obstacle in the replication or transcription process. Various studies have reported that when cells with DNA damage caused by TOP1cc enter mitotic phase, aberrant chromosome segregations happen, ultimately resulting in cell death [16,20]. Our data show that expression of DNA damage markers, such as γH2AX and p-RPA, were elevated and DNA fragmentation was increased both in HER2-positive cell lines and HER2-low cell lines after T-DXd treatment. Furthermore, T-DXd suppress G2/M cell cycle progression in HER2-positive cell lines and HER2-low cell lines. Additionally, T-DXd treatment induced nuclear anaplasia, in both HER2-positive and HER2-low cell lines. These morphological changes were also recapitulated in human breast cancer tissue after T-DXd treatment. These results suggest that T-DXd induces nuclear anaplasia through the accumulation of DNA damage by the payload. Interestingly, despite using a higher treatment dose for gastric cancer cell lines, the proportion of cells in the G2/M phase and the number of abnormal nuclei were significantly higher in breast cancer cell lines compared to gastric cancer cell lines after T-DXd treatment. This observation aligns with the in vitro antitumor efficacy of T-DXd and clinical data [10].

In the present study, we confirmed that T-DXd induce DNA damage as a monotherapy. Previous studies have shown a synergistic effect between DNA-damaging agents, including TOP1 inhibitor, and DNA damage response inhibitors [21-23]. Moreover, HRD tumors exhibit heightened susceptibility to TOP1-mediated DNA damage, suggesting that T-DXd could be particularly effective in HRD-driven malignancies [24]. Our findings thus contribute to the rationale for expanding the application of T-DXd into HRD or HRR mutated tumor types, and warrant further exploration of combination strategies.

Poly (ADP-ribose) polymerase-1 (PARP1) is a DNA damage sensor protein, which catalyzes the transfer ADP-ribose polymers (PAR polymers) onto itself and target proteins [25]. The DNA single-strand breaks (SSBs) occurred by TOP1cc are recognized by PARP1 and repaired by DNA damage repair molecules which are recruited by PARylation [23,24]. Several studies have reported that inhibition of PARP1 enhances the cytotoxicity of TOP1 inhibitors. PARP1 deficiency was shown to sensitize cells to camptothecin [24]. PARP inhibitors showed synergy with TOP1 inhibitors by inhibiting the repair of DNA damage caused by TOP1cc [24,25]. Concordantly these results, T-DXd combined with AZD5305, a selective inhibitor of PARP1, increased the DNA damage and cell death in vitro, and growth inhibition in vivo [26]. A phase 1/2a PETRA clinical trial (NCT04644068) is currently evaluating the combination of T-DXd with AZD5305.

During cell cycle progression, unrepaired SSBs caused by TOP1cc could convert to DSB, and the ataxia telangiectasia and RAD3-related protein (ATR)–CHK1 pathway is activated leading to HRR [11,16,20]. Recently, it has been reported that molecules involved in HRR are associated with sensitivity of TOP1 inhibitor. Triple-negative breast cancer with BRCAness and high SLFN11 expression showed higher response to TOP1 inhibitors [11,22]. Exatecan, a TOP1 inhibitor, induced cancer cell death in synergy with AZD6738, an ATR inhibitor [22]. In the present study, we confirmed that DNA damage accumulation ultimately lead to apoptotic cell death in SNU-601, a HER2-low cell line with RAD51C deficiency, and in Capan-1, a HER2-low cell line with BRCA2 mutation. T-DXd successfully inhibited tumor growth even in a low dose (4 mg/kg) in SNU-601 xenograft models. These data suggest that T-DXd might be effective in tumors with HRD, even when the HER2 expression is low.

We have previously shown the HRD-associated features of SNU-601, including γH2AX accumulation without RAD51 nuclear foci formation following DNA damage. RAD51C deficiency in this cell line has been shown to confer sensitivity to olaparib, which is reversible upon RAD51C reconstitution [27], supporting impaired HRR function. Potent efficacy of T-DXd in our in vivo SNU-601 xenograft models suggests the promising potential of the combination of T-DXd with targeted drugs which confer HRD, which warrants further investigation.

While these findings are promising and suggest potential for broader application, several limitations should be noted. First, our in vivo study relied on a single HER2-low cell line (SNU-601), which may not reflect the full diversity of HER2-low tumors. Second, we did not directly explore T-DXd in combination with DNA damage response inhibitors, although our data suggest potentially synergistic effects. Finally, the in vivo study was conducted using a single-dose regimen, which limits insights into potential long-term toxicity or repeated dosing effects. Addressing these issues in future research would further clarify the clinical applicability of T-DXd.

In conclusion, T-DXd showed potent cytotoxicity in human cancer cell lines with a wide range of HER2 expression. It induced DNA damage and nuclear anaplasia in HER2-positive and HER2-low cell lines, especially in those with HRD. These results suggest that T-DXd could be a novel treatment strategy for HRD tumors expressing HER2. These data could also provide a scientific ground for the clinical application of T-DXd in combination with targeted therapy which disrupts HRR pathway.

Electronic Supplementary Material

Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).

crt-2025-201_S1_Table.pdf

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NOTES

Ethical Statement

All animal experiments were conducted in the animal facility of Seoul National University under institutional guidelines and prior approval from the Institutional Animal Care and Use Committee (IACUC No.: 18-0252-S1A0). Use of human derived tissue samples was approved by the Institutional Review Board (IRB) of SNUH (IRB No.: 2310-165-1480), and the consent process was waived by IRB.

Author Contributions

Conceived and designed the analysis: Im SA, Lee KH, Koh J, Min A.

Performed the experiments: Kim SH, Park Y, Min A, Kim YJ, Ham S, Kim S.

Collected the data: Kim SH, Park Y, Ham S, Koh J.

Contributed data or analysis tools: Park HY, Ham S, Lee DW, Ryu HS, Koh J, Kim JS, Lee KH.

Performed the analysis: Kim SH, Park Y, Park HY, Ham S, Lee DW, Ryu HS, Koh J, Kim JS, Lee KH.

Wrote the paper: Kim SH, Park HY, Park Y, Ham S.

Supervision, manuscript editing: Koh J, Lee KH, Im SA.

Conflict of Interest

Seock-Ah Im is a recipient of research funds from AstraZeneca Inc., Boryung Pharm, Daiichi Sankyo Co., Ltd, Daewoong Pharm, Eisai, Pfizer, Roche outside of this laboratory works and has consultant and advisory role for AstraZeneca, Daiichi Sankyo Co., Ltd, Eisai, Hanmi Corp, MSD, Lilly, Novartis, Pfizer, and Roche. Jiwon Koh has advisory role for AstraZeneca outside of this laboratory works.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (IRIS No. RS-2024-00454656). Daiichi Sankyo Co., Ltd kindly provide trastuzumab deruxtecan (T-DXd; DS-8201a) used for this study under research proposal from Cancer Research Institute, Seoul National University (SNU 0431-20200010).

Fig. 1.

Antitumor effect of trastuzumab deruxtecan (T-DXd) in wide range of human epidermal growth factor receptor 2 (HER2) expressing human cancer cell lines. (A, B) The long-term growth inhibition effect of T-DXd were examined using a colony formation assay. Colony formation was determined by the number of colonies in controls were set to 100%. Values are expressed as mean±standard error (SE).

(C, D) Apoptotic cell death was analyzed by annexin V assay. Cells were treated with vehicle or T-DXd for 5 days. The percentages of annexin V–positive cells, indicating early and late apoptotic population was calculated. Bars represent as relative fold change of annexin V–positive population mean±SE. *p < 0.05, **p < 0.01, *** p < 0.001.

(E, F) Cells were treated with the indicated concentrations of T-DXd for 120 hours. Whole-cell lysates were analyzed for expression level of proteins by Western blot. Actin was measured as a loading control.

Fig. 2.

Internalization and human epidermal growth factor receptor 2 (HER2) signal transduction inhibitory effect of trastuzumab deruxtecan (T-DXd). (A, B) Cell surface binding and internalization of T-DXd. Internalization of T-DXd is inferred as the mean fluorescent intensity reduction of T-DXd bound to the cell surface. T-DXd is internalized over the time in HER2-positive cell lines (SK-BR-3, HCC-1419, NCI-N87, and SNU-216) and HER2-low cell lines (ZR-75-1, Capan-1, SNU-484, SNU-601, SNU-638, SNU-4257, and SNU-668). *p < 0.05, **p < 0.01, ***p < 0.001.

(C, D) Cells were treated with T-DXd for 120 hours. The expression of HER2 signal–mediated molecules was confirmed by western blotting.

Fig. 3.

Trastuzumab deruxtecan (T-DXd) induces DNA damage accumulation. Cells were treated with the indicated dose of T-DXd for 120 hours. (A, B) The expression of DNA damage response molecules were analyzed by western blot. (C, D) Alkaline comet assay conducted to identify DNA damage in cells. Scale bars=20 μm. At least 50 cells were counted for each experiment. Bars represent as relative fold change of tail moment mean±standard error. *p < 0.05, **p < 0.01, ***p < 0.001.

(E, F) RAD51 (green) and γH2AX (red) foci in cells were determined using confocal microscopy. DNA was counterstained with DAPI (blue). Scale bars=10 μm.

Fig. 4.

Cell cycle arrest in G2/M phase, and formation of aberrant nucleus upon trastuzumab deruxtecan (T-DXd) treatment. (A, B) The cell cycle distribution was analyzed by flow cytometry. The proportions of cells in G2/M phase are presented as a bar graph with standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001. (C, D) The expression level of G2/M phase transition regulation proteins was confirmed by western blotting.

(E, F) After T-DXd treatment for 120 hours, cell image was detected using confocal microscopy. Scale bars=10 μm. The percentages of abnormal nuclei were presented as a bar graph with standard error. **p < 0.01, ***p < 0.001. (G) Representative hematoxylin and eosin staining images of tumor sections before and after T-DXd treatment from a HER2-positive breast cancer patient are shown. Nuclear anaplastic features are more pronounced in the T-DXd–treated tumor, which are highlighted by arrows. Scale bars=50 μm.

Fig. 5.

Antitumor effect of trastuzumab deruxtecan (T-DXd) in human epidermal growth factor receptor 2–low expressing cell-derived xenograft model. Antitumor effect of T-DXd was assessed in the SNU-601 xenograft model. Mice were treated with indicated doses of T-DXd or vehicle via intravenous injection after the tumor volume reached 200 mm³. (A) The tumor volumes of each mouse were measured three times per week and are presented as a graph with standard error (SE). (B) To examine toxicity of T-DXd treatment, the body weight of each mouse was measured three times per week. Bars indicate SE. (C) Immunohistochemistry images of tumor section from mice treated with vehicle or T-DXd. After treatment, tumor tissues were resected from mice, and pathologic examinations were performed using hematoxylin and eosin (H&E) staining. To assess cell proliferation and apoptotic cell death, Ki-67 immunostaining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted. Scale bars=100 μm. (D) Ki-67 and TUNEL bar plot analysis. The percentages of Ki-67–positive cells, reflecting cell proliferation, and TUNEL-positive cells, indicating apoptotic cell death, were presented as a bar graph with SE. *p < 0.05, **p < 0.01, ***p < 0.001.

Table 1.

Characteristics of cell lines and IC₅₀ of T-DXd

Cancer type	Cell lines	ERBB2 gene status	HER2 IHC score	HRR status	IC₅₀ of T-DXd (ng/mL, mean±SE)	Classification
Breast	SK-BR-3	Amplified	Positive (3+)	WT	1.94±0.01	HER2-positive
	HCC1419	Amplified	Positive (3+)	WT	18.3±0.54	HER2-positive
	ZR-75-1	-	Ultralow	WT	30.25±3.96	HER2-ultralow
	MDA-MB-231	-	Negative (0)	WT	> 100	HER2-negative
Gastric	NCI-N87	Amplified	Positive (3+)	WT	15.3±0.16	HER2-positive
	SNU-216	Amplified	Positive (3+)	WT	102±2.49	HER2-positive
	SNU-484	-	Low (2+)	WT	219.8±24.2	HER2-low
	SNU-601	-	Low (1+)	RAD51C methylation	244.7±18.8	HER2-low
	SNU-638	-	Negative (0)	WT	> 500	HER2-low
	SNU-668	-	Negative (0)	WT	> 500	HER2-low
	SNU-4257	-	Negative (0)	WT	> 500	HER2-low
Pancreatic	Capan-1	-	Low (2+)	BRCA2 6174delT	143.8±33	HER2-low

HRR, homologous recombination repair; WT, wild type.

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REFERENCES

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Redefining HER2 in Breast Cancer: From Conventional Positivity to Low and Ultralow in the New Era of Antibody-Drug Conjugates
Jiwon Koh, Seock-Ah Im
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Case Report: Complete metabolic responses to trastuzumab–deruxtecan in HER2-altered solid tumors: two illustrative cases
Coline Le Meur, Pierre-Yves Le Roux, Pierre Alemany, Frédéric Chauvelot, Olivier Pradier, Clémence Niel, Alex Bellange, Christos Chouaid, Christophe Massard, Karim Amrane
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DNA Damage and Nuclear Anaplasia Induced by Trastuzumab Deruxtecan in Cancer Cells with Variable HER2 Expression and Homologous Recombination Deficiency Status

Cancer Res Treat. 2026;58(2):407-422. Published online August 4, 2025

DOI: https://doi.org/10.4143/crt.2025.201

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Abstract
Introduction
Materials and Methods
Results
Discussion
Electronic Supplementary Material
NOTES
REFERENCES

DNA Damage and Nuclear Anaplasia Induced by Trastuzumab Deruxtecan in Cancer Cells with Variable HER2 Expression and Homologous Recombination Deficiency Status

Fig. 1. Antitumor effect of trastuzumab deruxtecan (T-DXd) in wide range of human epidermal growth factor receptor 2 (HER2) expressing human cancer cell lines. (A, B) The long-term growth inhibition effect of T-DXd were examined using a colony formation assay. Colony formation was determined by the number of colonies in controls were set to 100%. Values are expressed as mean±standard error (SE). (C, D) Apoptotic cell death was analyzed by annexin V assay. Cells were treated with vehicle or T-DXd for 5 days. The percentages of annexin V–positive cells, indicating early and late apoptotic population was calculated. Bars represent as relative fold change of annexin V–positive population mean±SE. *p < 0.05, **p < 0.01, *** p < 0.001. (E, F) Cells were treated with the indicated concentrations of T-DXd for 120 hours. Whole-cell lysates were analyzed for expression level of proteins by Western blot. Actin was measured as a loading control.

Fig. 2. Internalization and human epidermal growth factor receptor 2 (HER2) signal transduction inhibitory effect of trastuzumab deruxtecan (T-DXd). (A, B) Cell surface binding and internalization of T-DXd. Internalization of T-DXd is inferred as the mean fluorescent intensity reduction of T-DXd bound to the cell surface. T-DXd is internalized over the time in HER2-positive cell lines (SK-BR-3, HCC-1419, NCI-N87, and SNU-216) and HER2-low cell lines (ZR-75-1, Capan-1, SNU-484, SNU-601, SNU-638, SNU-4257, and SNU-668). *p < 0.05, **p < 0.01, ***p < 0.001. (C, D) Cells were treated with T-DXd for 120 hours. The expression of HER2 signal–mediated molecules was confirmed by western blotting.

Fig. 3. Trastuzumab deruxtecan (T-DXd) induces DNA damage accumulation. Cells were treated with the indicated dose of T-DXd for 120 hours. (A, B) The expression of DNA damage response molecules were analyzed by western blot. (C, D) Alkaline comet assay conducted to identify DNA damage in cells. Scale bars=20 μm. At least 50 cells were counted for each experiment. Bars represent as relative fold change of tail moment mean±standard error. *p < 0.05, **p < 0.01, ***p < 0.001. (E, F) RAD51 (green) and γH2AX (red) foci in cells were determined using confocal microscopy. DNA was counterstained with DAPI (blue). Scale bars=10 μm.

Fig. 4. Cell cycle arrest in G2/M phase, and formation of aberrant nucleus upon trastuzumab deruxtecan (T-DXd) treatment. (A, B) The cell cycle distribution was analyzed by flow cytometry. The proportions of cells in G2/M phase are presented as a bar graph with standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001. (C, D) The expression level of G2/M phase transition regulation proteins was confirmed by western blotting. (E, F) After T-DXd treatment for 120 hours, cell image was detected using confocal microscopy. Scale bars=10 μm. The percentages of abnormal nuclei were presented as a bar graph with standard error. **p < 0.01, ***p < 0.001. (G) Representative hematoxylin and eosin staining images of tumor sections before and after T-DXd treatment from a HER2-positive breast cancer patient are shown. Nuclear anaplastic features are more pronounced in the T-DXd–treated tumor, which are highlighted by arrows. Scale bars=50 μm.

Fig. 5. Antitumor effect of trastuzumab deruxtecan (T-DXd) in human epidermal growth factor receptor 2–low expressing cell-derived xenograft model. Antitumor effect of T-DXd was assessed in the SNU-601 xenograft model. Mice were treated with indicated doses of T-DXd or vehicle via intravenous injection after the tumor volume reached 200 mm3. (A) The tumor volumes of each mouse were measured three times per week and are presented as a graph with standard error (SE). (B) To examine toxicity of T-DXd treatment, the body weight of each mouse was measured three times per week. Bars indicate SE. (C) Immunohistochemistry images of tumor section from mice treated with vehicle or T-DXd. After treatment, tumor tissues were resected from mice, and pathologic examinations were performed using hematoxylin and eosin (H&E) staining. To assess cell proliferation and apoptotic cell death, Ki-67 immunostaining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted. Scale bars=100 μm. (D) Ki-67 and TUNEL bar plot analysis. The percentages of Ki-67–positive cells, reflecting cell proliferation, and TUNEL-positive cells, indicating apoptotic cell death, were presented as a bar graph with SE. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

DNA Damage and Nuclear Anaplasia Induced by Trastuzumab Deruxtecan in Cancer Cells with Variable HER2 Expression and Homologous Recombination Deficiency Status

Cancer type	Cell lines	ERBB2 gene status	HER2 IHC score	HRR status	IC₅₀ of T-DXd (ng/mL, mean±SE)	Classification
Breast	SK-BR-3	Amplified	Positive (3+)	WT	1.94±0.01	HER2-positive
	HCC1419	Amplified	Positive (3+)	WT	18.3±0.54	HER2-positive
	ZR-75-1	-	Ultralow	WT	30.25±3.96	HER2-ultralow
	MDA-MB-231	-	Negative (0)	WT	> 100	HER2-negative
Gastric	NCI-N87	Amplified	Positive (3+)	WT	15.3±0.16	HER2-positive
	SNU-216	Amplified	Positive (3+)	WT	102±2.49	HER2-positive
	SNU-484	-	Low (2+)	WT	219.8±24.2	HER2-low
	SNU-601	-	Low (1+)	RAD51C methylation	244.7±18.8	HER2-low
	SNU-638	-	Negative (0)	WT	> 500	HER2-low
	SNU-668	-	Negative (0)	WT	> 500	HER2-low
	SNU-4257	-	Negative (0)	WT	> 500	HER2-low
Pancreatic	Capan-1	-	Low (2+)	BRCA2 6174delT	143.8±33	HER2-low

Table 1. Characteristics of cell lines and IC50 of T-DXd

HRR, homologous recombination repair; WT, wild type.