Differences in Responses to Neoadjuvant Anti-HER2 Therapy between HER2 2+/ISH+ and HER2 3+ in HER2-Positive Breast Cancer

Lingjun Ma; Ran Zheng; Lingyun Xu; Ying Zhu; Hong Yin; Xiaoqing Zhang; Rong Deng; Jue Wang; Xiaoming Zha

doi:10.4143/crt.2024.1200

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Original Article Breast cancer Differences in Responses to Neoadjuvant Anti-HER2 Therapy between HER2 2+/ISH+ and HER2 3+ in HER2-Positive Breast Cancer: Lingjun Ma¹, Ran Zheng¹, Lingyun Xu², Ying Zhu³, Hong Yin⁴, Xiaoqing Zhang⁵, Rong Deng³, Jue Wang¹, Xiaoming Zha¹; Cancer Research and Treatment : Official Journal of Korean Cancer Association 2026;58(2):501-512.
DOI: https://doi.org/10.4143/crt.2024.1200
Published online: April 21, 2025

¹Department of Breast Disease, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China

²Department of Breast Surgery, The Second People’s Hospital of Changzhou, The Third Affiliated Hospital of Nanjing Medical University, Changzhou Medical Center, Nanjing Medical University, Changzhou, China

³Department of General Surgery, Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, China

⁴Department of Breast Surgery, Women’s Hospital of Nanjing Medical University (Nanjing Women and Children’s Healthcare Hospital), Nanjing, China

⁵The Affiliated Hospital of Nanjing University of Chinese Medicine (Jiangsu Province Hospital of TCM), Nanjing, China

Correspondence: Rong Deng, Department of General Surgery, Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing 210009, China
Tel: 86-2583284725 E-mail: dengrong1977@163.com

Co-correspondence: Jue Wang, Department of Breast Disease, The First Affiliated Hospital of Nanjing Medical University, No. 300 Guangzhou Road, Nanjing 210000, China
Tel: 86-2568308171 E-mail: wangjue200011@njmu.edu.cn

Co-correspondence: Xiaoming Zha, Department of Breast Disease, The First Affiliated Hospital of Nanjing Medical University, No. 300 Guangzhou Road, Nanjing 210000, China
Tel: 86-2568308172 E-mail: njzhaxm@njmu.edu.cn

^*Lingjun Ma, Ran Zheng, and Lingyun Xu contributed equally to this work.

• Received: December 12, 2024 • Accepted: April 20, 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
Dual anti–human epidermal growth factor receptor 2 (HER2) drugs have become the standard regimen for neoadjuvant systemic treatment (NST) to HER2-positive breast cancer patients. However, the efficacy varies greatly among patients with different HER2 protein expression levels.
Materials and Methods
A total of 575 HER2-positive breast cancer patients from multiple centers throughout China from 2013 to 2022 were retrospectively analyzed. We compared clinicopathological features in different HER2 immunohistochemistry classes (HER2 2+/in situ hybridization [ISH] + or HER2 3+), and their difference in response to NST and survival with single or dual anti-HER2 drugs. Drug sensitivity assays were used to evaluate different efficacy of anti-HER2 drugs in vitro.
Results
Compared to HER2 3+ subgroup, the HER2 2+/ISH+ group had a higher proportion of hormone receptor–positive status (48.7% vs. 76.1%, p < 0.001), more HER2 protein loss after NST, lower pathological complete response (pCR) rate (46.07% vs. 16.24%, p < 0.001), and tended to have worse disease-free survival (DFS). In HER2 2+/ISH+ patients, treated with pertuzumab and trastuzumab in combination had no significant improvement in pCR (19.12% vs. 12.24%, p=0.287) and DFS (p=0.908) than using alone. Drug sensitivity assay showed poor efficacy with dual anti-HER2 drugs in HER2 2+/ISH+ cell lines; however, fam-trastuzumab deruxtecan drugs had a satisfactory effect.
Conclusion
Owing to the differences in clinicopathological features and treatment efficacy, we considered the HER2 2+/ISH+ group to be a distinct subtype and defined it as the HER2-moderate–positive subgroup. In this subgroup, dual anti-HER2 drugs did not exert significant improvement in pCR and DFS. Therefore, treatment optimization is warranted, with antibody-drug conjugate drugs as potential options.
Key words: Breast neoplasms, HER2-positive, Neoadjuvant targeted therapy, Pathologic complete response, Disease-free survival

Introduction

Around 15%-20% [1] of all breast cancer patients are human epidermal growth factor receptor 2 (HER2)-positive, which is defined mainly based on immunohistochemistry (IHC) detection of HER2 protein expression, including high (HER2 3+) and moderate expression (HER2 2+), with the latter requiring further fluorescence in situ hybridization (FISH) detection showing amplification on a genetic level (HER2 2+/in situ hybridization [ISH] +).

Various anti-HER2 drugs have been used as neoadjuvant and adjuvant therapy for HER2-positive patients. In fact, the NeoSphere [2] and PEONY [3] studies showed that dual anti-HER2 drugs, such as trastuzumab combined with pertuzumab, can produce higher pathological complete response (pCR) rates than single drug, making dual anti-HER2 therapy the standard regimen. However, emerging antibody-drug conjugate (ADC) drugs have shown good performance not only in HER2-positive patients but also in HER2 low-positive (HER2 2+/ISH–) [4] and ultra-low-positive (HER2 1+) [5] patients. This suggests that the conventional dichotomization of HER2 expression, namely HER2 positivity and negativity, lacks sufficient precision to provide guidance to anti-HER2 therapy, considering the substantial heterogeneity in HER2 protein levels.

Research on HER2-positive patients has also shown considerable heterogeneity in the efficacy of neoadjuvant anti-HER2 therapy. Several studies [6-12] have shown that HER2 2+/ISH+ patients have a significantly worse response to neoadjuvant systemic treatment (NST) than HER2 3+ patients. However, no study has yet clarified whether such a difference in efficacy stems from differences in protein expression or the underuse of targeted drugs. First, it remains controversial whether patients in the HER2 2+/ISH+ group with moderate HER2 protein expression have distinctive biological features that differentiate them from the HER2 3+ group [6-11], with data from relevant studies being scarce. Second, few studies have determined the correlation between the use of targeted drugs and HER2 protein expression level to analyze NST efficacy. For example, a study by Chen et al. [13] compared the pCR rates of HER2 2+/ISH+ and HER2 3+ patients who received neoadjuvant dual anti-HER2 therapy, whereas Cha et al. [14] compared the pCR rates with no, single, and dual neoadjuvant targeted drugs. However, neither of these studies investigated the differences in the response to and prognosis of anti-HER2 therapy between the two groups. Therefore, we questioned whether HER2 2+/ISH+ could be a subtype independent of HER2 3+ and whether there is any room to optimize the NST regimen for HER2 2+/ISH+ patients.

The current study analyzed the clinicopathological features and compared the efficacy of NST between HER2 3+ and HER2 2+/ISH+ patients. Finally, a new strategy for optimizing targeted therapy was preliminarily explored through drug-sensitive assay in vitro.

Materials and Methods

1. Patients

A total of 636 HER2-positive breast cancer patients who received NST containing targeted therapy and underwent surgery at four breast cancer centers throughout Nanjing, China from 2013 to 2022 were included. Patients who had distant metastasis at diagnosis, had a history of malignancy, had bilateral breast cancer, were lactating or pregnant, did not receive anti-HER2 neoadjuvant therapy or radical operation, and had a follow-up period of less than 12 months were excluded from the study. After excluding 61 patients, 575 HER2-positive patients were finally included (S1 Fig.).

2. IHC criteria

Hormone receptor (HR)–positive was defined as estrogen receptor positive and/or progesterone receptor positive, which are both defined as no less than 1% of the invasive tumor cells staining positive via IHC. According to the 2018 ASCO/CAP (American Society of Clinical Oncology/College of American Pathologists) HER2 testing guideline [15], HER2-positive was defined as HER2 3+ and HER2 2+ by IHC with the presence of HER2 amplification by FISH (ISH+). A high Ki-67 level was defined as a Ki-67–positive cell ratio > 20% according to the expert consensus of St. Gallen in 2021.

3. Treatment

All patients received targeted therapy containing trastuzumab (Herceptin) with or without pertuzumab (Perjeta) and chemotherapy regimens mainly based on taxane and anthracycline. Details regarding the NST regimen are presented in S2 Fig. In the postoperative intensive adjuvant phase, all patients received targeted therapy or an ADC for 1 year. The decision to administer radiotherapy was made by the radiation oncologists.

4. NST efficacy evaluation

pCR was defined as the disappearance of invasive cancer in the breast, regardless of ductal carcinoma in situ, as well as the absence of invasive cancer in the resected axillary tissue. All patients were included in follow-up schedule, with the last follow-up having been conducted in August 2024. Disease-free survival (DFS) was defined as the interval between the time of surgery and tumor recurrence, metastasis, tumor-related disease, or the date of censoring.

5. Cell lines and cell counting kit 8

Human breast cancer cell lines BT474, SKBR3, and HCC1569 were kindly gifted by Dr. Ziyi Fu, whereas MDA-MB-361 was purchased from Pricella Life Science & Technology Co., Ltd. The cell lines were cultured according to the recommended specifications. According to IHC and FISH results reported in the literature [16], BT474 and SKBR3 are human breast cancer cell lines with HER2 3+, whereas HCC1569 and MDA-MB-361 are HER2 2+/ISH+ cell lines.

Cells were incubated in 96-well plates at a density of 5,000 cells and treated with trastuzumab, pertuzumab, or fam-trastuzumab deruxtecan (T-Dxd) (Enhertu) for 120 hours. Cell viability was determined using the cell counting kit 8 reagent and cultured at 37°C for 1 hour. Pro-11 Multiskan FC (Thermo Fisher) was used to examine optical density of each well at 450 nm.

6. Statistical analysis

The chi-square test (χ²) was used to compare categorical variables, whereas the Cox-regression model was applied to analyze the independent influence factor of pCR. Category changes in HER2 IHC classes were analyzed using the concordance rate and Kappa test and was graphically reported using Sankey diagrams. The probability of DFS was estimated using the Kaplan-Meier method and the log-rank test. During our analyses, two-sided p-values of < 0.05 indicated statistical significance. All statistical analyses were conducted using SPSS ver. 26.0 software (IBM Corp.) and GraphPad Prism ver. 10.1.2 (GraphPad Software).

Results

1. Clinicopathological characteristics of the HER2-positive patients

This study included a total of 575 HER2-positive patients, containing 117 (20.3%) HER2 2+/ISH+ and 458 (79.7%) HER2 3+ patients (S1 Fig.).

Table 1 summarizes the clinical and pathological characteristics according to HER2 IHC classes. No significant difference in most clinical characteristics at baseline were observed between the HER2 2+/ISH+ and HER2 3+ groups. However, significantly more HER2 2+/ISH+ patients than HER2 3+ patients were HR-positive (76.1% vs. 48.7%, p < 0.001). In terms of NST regimens, significantly fewer HER2 2+/ISH+ patients than HER2 3+ patients received dual anti-HER2 therapy (58.1% vs. 74.0%, p < 0.001).

2. Category change in HER2 IHC class from core needle biopsy pre-NST to residual disease post-NST

A total of 309 patients did not achieve pCR and underwent HER2 IHC retesting on surgical pathology specimens. Changes in HER2 expression determined through IHC were analyzed on paired samples from patients with available pre-NST core needle biopsy samples and post-NST surgical excision samples (Fig. 1). Among 89 patients in the initial HER2 2+/ISH+ group, 50 remained HER2 2+ after NST (56.2%), 21 became HER2 3+ (23.6%), and 18 patients became HER2 1+ or zero expression (22.2%). In contrast, among 220 patients in the initial HER2 3+, 157 remained HER2 3+ (71.4%) after NST, 55 became HER2 2+ (25.0%), and only eight became HER2 1+ or zero (3.7%). The overall inconsistency rate was 33.31% (kappa=0.329, p < 0.001) (Fig. 1).

3. Response to NST and survival analysis according to HER2 IHC status

Before NST, both groups had similar mean values for the maximum tumor diameter evaluated via ultrasound (p=0.423) (Table 1). After NST, the pathological results showed that compared to the HER2 3+ group, the HER2 2+/ISH+ group had a significantly increased mean value for the largest diameter of the invasive tumor (7.77 vs. 12.78 mm, respectively; p=0.001) and number of positive axillary lymph nodes (0.98 vs. 2.35, respectively; p=0.001) but decreased Miller Payne level (p < 0.001) (Table 1).

A comparison of the pCR rate showed that HER2 2+/ISH+ patients had a significantly lower pCR rate than had the HER2 3+ group (16.24% vs. 46.07%, respectively; p < 0.001) (Fig. 2). Subgroup analyses showed that pCR was significantly lower in HER2 2+/ISH+ patients than in HER2 3+ patients in both the HR+ and HR– subgroups (p < 0.001 vs. p=0.041, respectively), as well as in the single or dual anti-HER2 therapy subgroup (p=0.024 and p < 0.001, respectively) (S3 Fig.). Baseline characteristics of HER2 2+/ISH+ and HER2 3+ patients were compared in HR and anti-HER2 therapy subgroups respectively, no difference was found in all subgroups (S4 Table).

In terms of DFS, the median follow-up was 49.6 months for all patients (n=575). Kaplan-Meier analysis showed that DFS was significantly better in patients who obtained pCR than in those who did not (p < 0.001) (S5A Fig.). Stratified analysis of HER2 IHC classes showed a trend toward shorter DFS in the HER2 2+/ISH+ group than in the HER2 IHC 3+ group (estimated 5-year DFS, 83.6% vs. 90.6%), although the difference was not significant (p=0.222) (Fig. 3). Subgroup analyses of HR status showed no significant difference in DFS (estimated 5-year DFS, 89.3% vs. 88.4%; p=0.255) (S5B Fig.). Regarding anti-HER2 therapy, DFS was worse with single than with dual anti-HER2 drugs (estimated 5-year DFS, 85.4% vs. 92.6%; p=0.038) (S5C Fig.).

Based on the aforementioned results, we believe that significant differences in HR expression, HER2 expression stability, and NST efficacy exist between HER2 2+/ISH+ and HER2 3+ patients. Thus, we considered HER2 2+/ISH+ patients as a distinct subtype independent from HER2 3+ patients and defined them as the HER2-moderate–positive (HER2-mod) subgroup.

4. NST efficacy with different anti-HER2 therapies among HER2-mod patients

(1) Pathological complete response

We further analyzed the factors influencing pCR in the HER2-mod subgroup. Factors deemed significant (p < 0.15) on univariate regression analysis, as well as a few recognized factors influencing the pCR rate, were included in the multivariate regression analysis (S6 Table). Notably, our results showed that the use of trastuzumab alone or combined with pertuzumab had no significant effect on pCR (odds ratio [OR], 0.537; 95% confidence interval [CI], 0.171 to 1.685; p=0.287) (Fig. 4A), with the pCR rates being 12.24% and 19.12%, respectively (S7 Fig.). However, menstrual status and HR status were found to be independent factors for pCR (p=0.025 and p=0.014, respectively) (Fig. 4A).

(2) Disease-free survival

In HER2-mod patients, the median follow-up duration was 46 months (n=117). Kaplan-Meier analysis showed no significant difference in DFS with single or dual anti-HER2 therapy (estimated 5-year DFS, 83.0% vs. 87.3%; p=0.908) (Fig. 5A).

5. Response to different anti-HER2 therapies among HER2 3+ patients

(1) Pathological complete response

Different from HER2-mod group, multivariate regression analysis in the HER2 3+ group showed that the use of single or dual anti-HER2 therapy independently influenced pCR. HER2 3+ patients responded significantly worse to single anti-HER2 therapy than to dual anti-HER2 therapy (p < 0.001; OR, 0.362; 95% CI, 0.228 to 0.577) (Fig. 4B, S8 Table). The pCR rates were 28.57% and 52.21%, respectively (S7 Fig.). HR status had no significant effect on pCR in the HER2-mod group, in contrast to the HER2 3+ group (p=0.105) (Fig. 4B).

(2) Disease-free survival

The median follow-up duration was 40 months (n=458). Kaplan-Meier analysis showed that DFS was significantly worse with single than with dual anti-HER2 drugs (estimated 5-year DFS, 86.3% vs. 93.2%; p=0.027) (Fig. 5B).

6. In vitro sensitivity of HER2-positive breast cancer cells to anti-HER2 agents

To explore the potential optimized regimens for HER2-mod patients, we studied a panel of four HER2-overexpressing breast cancer cell lines previously determined as HER2 3+ (SK-BR3, BT-474) or HER2 2+/ISH+ (MDA-MB-361, HCC1569) [16]. The in vitro growth inhibition assay showed that when treated with trastuzumab (Fig. 6A), pertuzumab (Fig. 6B), and in combination (Fig. 6C), both HER2-mod cell lines, MDA-MB-361 and HCC1569, showed significantly less tumor growth inhibition than HER2 3+ cells. However, treatment with T-Dxd induced high growth inhibition activity in all four cell lines (Fig. 6D).

In the HER2-mod cell lines, combining trastuzumab and pertuzumab did not promote a significant increase in efficacy when compared to using them individually, whereas T-Dxd demonstrated the strongest growth inhibition (Fig. 7A and B). In contrast, in the HER2 3+ cell lines, dual anti-HER2 drugs showed better response than single anti-HER2 drugs (Fig. 7C), with the efficacy being even better with T-Dxd (Fig. 7D).

Discussion

1. HER2-mod type differs from HER2 3+ type in pathological and clinical features

(1) HER2 protein expression

The most essential difference between the HER2-mod and HER2 3+ groups in this study was that for HER2 protein expression levels. Previous studies have shown that differences in HER2 protein expression and/or gene amplification levels significantly affect the efficacy of anti-HER2 therapy [2,12]. Interestingly, the N9831 study [17] found that trastuzumab had no benefit in terms of DFS in HER2 IHC-ISH+ patients, and no difference in DFS was observed according to ISH+ and ISH− status among HER2 3+ patients, suggesting that protein expression may be a more critical influencing factor than gene expression. Together with the fact that commonly used anti-HER2 drugs mainly target the HER2 protein rather than its gene, we believe that HER2 protein expression is the key driver, which explains why HER2 protein expression was used as the grouping criterion in this study.

(2) HR status

A crosstalk is present between the HR and HER2 signaling pathways at the molecular level, and their expressions are negatively correlated [18,19]. Our data showed that significantly more patients in the HER2-mod group than in the HER2 3+ were HR+, which is consistent with the results reported in other studies [7,11]. This finding directly facilitated the use of different treatment options, that is, more patients in the HER2-mod group received endocrine therapy. This is also one of the important differences between HER2-mod and HER2 3+ at the pathological level.

(3) Change in HER2 IHC class

Studies had reported that HER2 protein loss after NST was significantly higher in HER2-mod than in HER2 3+ patients [3,20]. This finding had also been confirmed in the current study, which suggests that the HER2 protein was not only less expressed but also less stable in the HER2-mod group.

(4) pCR rate and residual disease

The pCR rate in the HER2-mod group in the current study was significantly lower than that in the HER2 3+ group. Several other studies have also reported the same results [6-11]. Among non-pCR patients, our analyses revealed that several pathological features of residual disease, namely maximum tumor diameter, number of metastatic lymph nodes, and Miller Payne level, were significantly worse in the HER2-mod group than in the HER2 3+ group. These findings showed significant differences in pathological features and clinical efficacy between HER2-mod and HER2 3+ patients.

(5) Survival

In terms of survival, several studies [17,20,21] have concluded a difference in prognosis between the two groups. In the current study, however, the DFS was numerically worse in the HER2-mod group than in the HER2 3+ group, although not significantly. Some possible reasons could be the relatively small sample size (n=117) considering the naturally low proportion of HER2-mod in HER2-positive breast cancer patient, resulting in reduced statistical power. In addition, the late introduction of pertuzumab into China (December 2018) resulted in a shorter follow-up duration in the dual anti-HER2 therapy group (median follow-up duration, dual-targeted vs. single-targeted group, 33 vs. 71 months), which may have somewhat also affected the results of the survival analysis.

Based on the presented evidence on the differences in multiple pathological and clinical features and treatment response, we hypothesize that HER2-mod may be a separate subtype that distinguishes itself from the HER2 3+ group. Thus, targeted therapy and its efficacy in the HER2-mod subgroup deserve further analysis.

2. Dual anti-HER2 therapy did not significantly improve pCR and DFS in the HER2-mod group

Our findings showed that among HER2 3+ patients, those using trastuzumab and pertuzumab together had a significantly higher pCR rate and DFS than those using trastuzumab alone, which was consistent with the results of the NeoSphere [2] and PEONY [3] studies.

However, only a few studies have focused on HER2-mod patients. The NeoSphere study [22] showed that neoadjuvant trastuzumab and pertuzumab did not improve pCR in the low HER2 mRNA group (lower than median). However, no comparison was performed at the protein level. A study by Katayama et al. [23] found pCR rates of 19% and 18% with single and dual anti-HER2 drugs in the HER2-mod group, which were numerically similar; however, no statistical comparison was made. Therefore, in the HER2-mod group, the difference in efficacy between using single and dual anti-HER2 drugs remains unclear. Our study showed that in the HER2-mod group, dual anti-HER2 drugs did not promote increased pCR or DFS rates relative to single anti-HER2 drugs. This finding tentatively suggests that the addition of pertuzumab to neoadjuvant trastuzumab did not improve efficacy in the HER2-mod group.

Given that the current standard regimen involves neoadjuvant trastuzumab and pertuzumab, approval for clinical prospective studies comparing the efficacy of single and dual anti-HER2 drugs in the HER2-mod group would be difficult. Hence, we performed experiments at the cytological level. Drug sensitivity assays showed that using trastuzumab together with pertuzumab did not increase growth inhibition activity in HER2-mod cell lines compared to using them individually but did promote a significant increase in growth inhibition activity in HER2 3+ cell lines. These in vitro experiments provide additional evidence further supporting HER2-mod as a distinct subgroup.

3. Optimizing the NST regimen for HER2-mod patients

When the efficacy of trastuzumab is poor, clinicians usually employ two strategies to increase the efficacy: one is to combine drugs with different mechanisms and the other is to use ADC drugs that are based on the modification of trastuzumab, such as T-DM1 or T-Dxd. Theoretically, trastuzumab and pertuzumab can bind different structural domains of HER2, thereby exerting synergistic effects [24]. In essence, however, both drugs still target the HER2 protein. The relatively low expression of the HER2 protein in the HER2-mod group may mainly explain the poor response to dual drugs. In contrast, the efficacy of ADC drugs is disproportional to the HER2 protein expression [11], they exhibit good therapeutic effects even in HER2-low patients [4]. The reason is that ADC drugs target HER2 to facilitate internalization of the cytotoxic agent into HER2-expressing cells and have a bystander effect additionally. Therefore, we believe that ADC drugs have the potential to improve the NST efficacy in the HER2-mod group.

To date, direct clinical evidence for the use of ADC among HER2-mod patients in NST is still lacking. The DESTINY-Breast 11 trial [25], which applied T-Dxd to early-stage HER2-positive breast cancer patients, is still ongoing. Therefore, we decided to perform exploratory in vitro experiments. Our results showed that although HER2-mod cells responded poorly to trastuzumab, pertuzumab, and their combination, they responded well to T-Dxd. Similar results have been reported in a study [26], wherein colon cancer cell lines whose HER2 protein expression was lower than the median, responded poorly to trastuzumab and pertuzumab but were sensitive to T-DM1 and T-Dxd. Similar results were also reached in vivo in a study by Barok [27], wherein JIMT-1 (HER2-mod, trastuzumab-resistant breast cancer cell) was used in the cell line-derived xenograft assay, and T-DM1 had the ability to inhibit tumor growth.

All preliminary results presented earlier highlight the feasibility of ADC drugs to optimize the NST regimen for HER2-mod patients. Further in vivo experiments and prospective clinical trials are still warranted. Ongoing trials, such as DESTINY-Breast 11 [25], SHAMROCK [28], and ARIADNE [29], which apply T-Dxd to NST to HER2-positive patients, are expected to provide more clinical evidence.

This study has some limitations worth noting. First, given that this clinical study was retrospective in nature, the enrollment may be biased. The long-time span of enrollment (2013-2022) and the time pertuzumab launched in China (December 2018) led to different rates of dual anti-HER2 therapy in HER2-mod and HER2 3+ patients. Second, the proportion of HER2-mod patients among HER2-positive patients is naturally low, leading to a relatively small sample size. Moreover, the short follow-up period may have affected our survival analyses. In addition, no ISH data were available for post-NST specimens in this study. This is because once patients are tested HER2+ before NST, they will still continue anti-HER2 therapy even if they become HER2 2+/ISH– after surgery. Furthermore, FISH testing is not routinely performed again on post-NST specimens given the high cost of testing in this region.

In summary, HER2-mod breast cancer varies significantly from HER2 3+ breast cancer in terms of pathological features of the tumor, clinical efficacy of anti-HER2 drugs, treatment strategies, and drug sensitivity in vitro, suggesting that it may be a separate subtype. Conventional anti-HER2 drugs like trastuzumab and pertuzumab did not exhibit good efficacy for the treatment of the HER2-mod subtype even when used in combination, with ADC drugs being expected to provide a better solution.

Electronic Supplementary Material

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

crt-2024-1200_S1_Fig.pdf

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crt-2024-1200_S4_Table.pdf

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crt-2024-1200_S8_Table.pdf

NOTES

Ethical Statement

The study was conducted in strict compliance with the principles of the Declaration of Helsinki, and was approved by Ethics Review Board of the First Affiliated Hospital of Nanjing Medical University in September 2024 (2024-SR-768). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Author Contributions

Conceived and designed the analysis: Ma L, Deng R, Wang J, Zha X.

Collected the data: Ma L, Zheng R, Xu L, Zhu Y, Yin H, Zhang X.

Contributed data or analysis tools: Xu L, Zhu Y, Yin H, Zhang X, Zha X.

Performed the analysis: Ma L, Zheng R, Xu L, Deng R.

Wrote the paper: Ma L, Wang J, Zha X.

Conflicts of Interest

Conflict of interest relevant to this article was not reported.

Funding

This research was partially supported by the Chinese Society of Clinical Oncology Foundation (Y-HR2020MS-0421); Jiangsu Province Elderly Health Research Project (LKM2022033, LKM2023023); Wu Jieping Medical Foundation Special Fund for Clinical Research (320.6750.2023-11-29); and China Breast Surgery Young Physician Research Award Advantage Support Program (2020-CHPASLP-01).

Acknowledgments

We gratefully acknowledge all patients, doctors and nurses for their contribution to this study.

Fig. 1.

Category change of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) from core needle biopsy pre–neoadjuvant systematic treatment (NST) to residual disease post-NST. ISH, in situ hybridization.

Fig. 2.

The pathological complete response (pCR) rate by human epidermal growth factor receptor 2 (HER2) status. ISH, in situ hybridization.

Fig. 3.

Subgroup analysis of disease-free survival according to human epidermal growth factor receptor 2 (HER2) status. ISH, in situ hybridization.

Fig. 4.

Multivariate regression analysis of pathological complete response (pCR) in human epidermal growth factor receptor 2 (HER2)-moderate–positive (HER2-mod) and HER2 3+ groups. (A) HER2-mod group. (B) HER2 3+ group. CI, confidence interval; cN, clinical node staging; cT, clinical tumor staging; HR, hormone receptor; OR, odds ratio.

Fig. 5.

Survival analysis of disease-free survival stratified by anti–human epidermal growth factor receptor 2 (HER2) therapy in HER2-moderate–positive (HER2-mod) and HER2 3+ groups. (A) HER2-mod group. (B) HER2 3+ group.

Fig. 6.

In vitro sensitivity of human epidermal growth factor receptor 2 (HER2)–positive breast cancer cells to anti-HER2 agents. (A) Trastuzumab. (B) Pertuzumab. (C) Pertuzumab (100 μg/mL)+trastuzumab. (D) T-Dxd (fam-trastuzumab deruxtecan). HER2-mod, HER2 moderate expression.

Fig. 7.

In vitro growth inhibition assay of different anti–human epidermal growth factor receptor 2 (HER2) agents to HER2-positive breast cancer cells. (A) MDA-MB-361 (HER2-mod). (B) HCC1569 (HER2-mod). (C) SK-BR3 (HER2 3+). (D) BT474 (HER2 3+). HER2-mod, HER2 moderate expression; T-Dxd, fam-trastuzumab deruxtecan.

Table 1.

Clinicopathological features of different HER2+ classes

Characteristic	Total (n=575)	HER2 status		χ²	p-value
Characteristic	Total (n=575)	HER2 2+/ISH+ (n=117)	HER2 3+ (n=458)	χ²	p-value
Age at diagnosis (yr)	50.46±9.63	49.15±10.85	50.79±9.28	-	0.134
Menopausal status
Premenopausal	283 (49.2)	62 (53.0)	221 (48.3)	0.837	0.360
Postmenopausal	292 (50.8)	55 (47.0)	237 (51.7)
Tumor location
Left	298 (51.8)	60 (51.3)	238 (52.0)	0.017	0.895
Right	277 (48.2)	57 (48.7)	220 (48.0)
Clinical tumor staging
cT1	33 (5.8)	6 (5.2)	27 (5.9)	4.972	0.174
cT2	330 (57.6)	71 (61.2)	259 (56.7)
cT3	92 (16.1)	23 (19.8)	69 (15.1)
cT4	118 (20.6)	16 (13.8)	102 (22.3)
Clinical node staging
cN0	74 (12.9)	12 (10.3)	62 (13.5)	5.234	0.177
cN1	212 (36.9)	46 (39.3)	166 (36.2)
cN2	205 (35.7)	48 (41.0)	157 (34.3)
cN3	84 (14.6)	11 (9.4)	73 (15.9)
HR status
Negative	263 (45.7)	28 (23.9)	235 (51.3)	28.147	< 0.001
Positive	312 (54.3)	89 (76.1)	223 (48.7)
Ki-67 expression (%)
≤ 20	20 (3.5)	5 (4.3)	15 (3.3)	0.268	0.605
> 20	553 (96.5)	112 (95.7)	441 (96.7)
Neoadjuvant systemic therapy
Chemotherapy+single anti-HER2 therapy	168 (29.2)	49 (41.9)	119 (26.0)	11.389	0.001
Chemotherapy+dual anti-HER2 therapy	407 (70.8)	68 (58.1)	339 (74.0)
Maximum diameter of tumor before NST (mm)	36.96±14.40	37.96±14.45	36.71±14.40		0.423
Maximum diameter of tumor after NST (mm)	17.64±13.02	19.50±13.88	17.11±12.75		0.223
Radiological response rate of breast tumor (%)	52.56±32.48	49.57±30.38	53.46±33.11		0.429
Breast surgery
Mastectomy surgery	512 (89.0)	109 (93.2)	403 (88.0)	2.554	0.110
Breast-conserving surgery	63 (11.0)	8 (6.8)	55 (12.0)
Axillary surgery
ALND	523 (91.0)	110 (94.0)	413 (90.2)	1.673	0.196
SLNB	52 (9.0)	7 (6.0)	45 (9.8)
Pathological tumor size (mm)	8.79±13.78	12.78±12.48	7.77±13.92		0.001
No. of positive nodes after surgery	1.29±2.79	2.35±3.62	0.98±2.42		0.001
Miller Payne level
1	15 (2.7)	7 (6.3)	8 (1.8)	22.817	< 0.001
2	69 (12.2)	21 (18.8)	48 (10.6)
3	122 (21.6)	34 (30.4)	88 (19.5)
4	106 (18.8)	16 (14.3)	90 (19.9)
5	252 (44.7)	34 (30.4)	218 (48.2)

Values are presented as mean±SD or number (%). ALND, axillary lymph node dissection; HER2, human epidermal growth factor receptor 2; HR, hormone receptor; ISH, in situ hybridization; NST, neoadjuvant systematic treatment; SD, standard deviation; SLNB, sentinel lymph node biopsy.

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Differences in Responses to Neoadjuvant Anti-HER2 Therapy between HER2 2+/ISH+ and HER2 3+ in HER2-Positive Breast Cancer

Cancer Res Treat. 2026;58(2):501-512. Published online April 21, 2025

DOI: https://doi.org/10.4143/crt.2024.1200

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

Differences in Responses to Neoadjuvant Anti-HER2 Therapy between HER2 2+/ISH+ and HER2 3+ in HER2-Positive Breast Cancer

Fig. 1. Category change of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) from core needle biopsy pre–neoadjuvant systematic treatment (NST) to residual disease post-NST. ISH, in situ hybridization.

Fig. 2. The pathological complete response (pCR) rate by human epidermal growth factor receptor 2 (HER2) status. ISH, in situ hybridization.

Fig. 3. Subgroup analysis of disease-free survival according to human epidermal growth factor receptor 2 (HER2) status. ISH, in situ hybridization.

Fig. 4. Multivariate regression analysis of pathological complete response (pCR) in human epidermal growth factor receptor 2 (HER2)-moderate–positive (HER2-mod) and HER2 3+ groups. (A) HER2-mod group. (B) HER2 3+ group. CI, confidence interval; cN, clinical node staging; cT, clinical tumor staging; HR, hormone receptor; OR, odds ratio.

Fig. 5. Survival analysis of disease-free survival stratified by anti–human epidermal growth factor receptor 2 (HER2) therapy in HER2-moderate–positive (HER2-mod) and HER2 3+ groups. (A) HER2-mod group. (B) HER2 3+ group.

Fig. 6. In vitro sensitivity of human epidermal growth factor receptor 2 (HER2)–positive breast cancer cells to anti-HER2 agents. (A) Trastuzumab. (B) Pertuzumab. (C) Pertuzumab (100 μg/mL)+trastuzumab. (D) T-Dxd (fam-trastuzumab deruxtecan). HER2-mod, HER2 moderate expression.

Fig. 7. In vitro growth inhibition assay of different anti–human epidermal growth factor receptor 2 (HER2) agents to HER2-positive breast cancer cells. (A) MDA-MB-361 (HER2-mod). (B) HCC1569 (HER2-mod). (C) SK-BR3 (HER2 3+). (D) BT474 (HER2 3+). HER2-mod, HER2 moderate expression; T-Dxd, fam-trastuzumab deruxtecan.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Differences in Responses to Neoadjuvant Anti-HER2 Therapy between HER2 2+/ISH+ and HER2 3+ in HER2-Positive Breast Cancer

Characteristic	Total (n=575)	HER2 status		χ²	p-value
Characteristic	Total (n=575)	HER2 2+/ISH+ (n=117)	HER2 3+ (n=458)	χ²	p-value
Age at diagnosis (yr)	50.46±9.63	49.15±10.85	50.79±9.28	-	0.134
Menopausal status
Premenopausal	283 (49.2)	62 (53.0)	221 (48.3)	0.837	0.360
Postmenopausal	292 (50.8)	55 (47.0)	237 (51.7)
Tumor location
Left	298 (51.8)	60 (51.3)	238 (52.0)	0.017	0.895
Right	277 (48.2)	57 (48.7)	220 (48.0)
Clinical tumor staging
cT1	33 (5.8)	6 (5.2)	27 (5.9)	4.972	0.174
cT2	330 (57.6)	71 (61.2)	259 (56.7)
cT3	92 (16.1)	23 (19.8)	69 (15.1)
cT4	118 (20.6)	16 (13.8)	102 (22.3)
Clinical node staging
cN0	74 (12.9)	12 (10.3)	62 (13.5)	5.234	0.177
cN1	212 (36.9)	46 (39.3)	166 (36.2)
cN2	205 (35.7)	48 (41.0)	157 (34.3)
cN3	84 (14.6)	11 (9.4)	73 (15.9)
HR status
Negative	263 (45.7)	28 (23.9)	235 (51.3)	28.147	< 0.001
Positive	312 (54.3)	89 (76.1)	223 (48.7)
Ki-67 expression (%)
≤ 20	20 (3.5)	5 (4.3)	15 (3.3)	0.268	0.605
> 20	553 (96.5)	112 (95.7)	441 (96.7)
Neoadjuvant systemic therapy
Chemotherapy+single anti-HER2 therapy	168 (29.2)	49 (41.9)	119 (26.0)	11.389	0.001
Chemotherapy+dual anti-HER2 therapy	407 (70.8)	68 (58.1)	339 (74.0)
Maximum diameter of tumor before NST (mm)	36.96±14.40	37.96±14.45	36.71±14.40		0.423
Maximum diameter of tumor after NST (mm)	17.64±13.02	19.50±13.88	17.11±12.75		0.223
Radiological response rate of breast tumor (%)	52.56±32.48	49.57±30.38	53.46±33.11		0.429
Breast surgery
Mastectomy surgery	512 (89.0)	109 (93.2)	403 (88.0)	2.554	0.110
Breast-conserving surgery	63 (11.0)	8 (6.8)	55 (12.0)
Axillary surgery
ALND	523 (91.0)	110 (94.0)	413 (90.2)	1.673	0.196
SLNB	52 (9.0)	7 (6.0)	45 (9.8)
Pathological tumor size (mm)	8.79±13.78	12.78±12.48	7.77±13.92		0.001
No. of positive nodes after surgery	1.29±2.79	2.35±3.62	0.98±2.42		0.001
Miller Payne level
1	15 (2.7)	7 (6.3)	8 (1.8)	22.817	< 0.001
2	69 (12.2)	21 (18.8)	48 (10.6)
3	122 (21.6)	34 (30.4)	88 (19.5)
4	106 (18.8)	16 (14.3)	90 (19.9)
5	252 (44.7)	34 (30.4)	218 (48.2)

Table 1. Clinicopathological features of different HER2+ classes