TNBC, characterized by the lack of expression of hormone receptors and human epidermal growth factor receptor 2 (HER2), is known to have the worst outcomes among all breast cancer subtypes (1-3). Due to the absence of targetable receptors, systemic treatment for TNBC primarily relies on chemotherapy. The standard neoadjuvant chemotherapy (NAC) regimen for TNBC has traditionally consisted of cytotoxic agents, such as anthracyclines and taxanes, and it is well established that patients who achieve a pathological complete response (pCR) following NAC have a more favorable prognosis (4). In recent years, novel therapeutic strategies for TNBC based on tumor characteristics have emerged, and immune checkpoint inhibitor (ICI)-based treatments have been incorporated into NAC regimens for TNBC (5).

The recent success of trastuzumab deruxtecan, an antibody-drug conjugate targeting HER2, in treating HER2-low metastatic breast cancer has brought renewed attention to HER2-low tumors (6). Meanwhile, the biological significance of HER2 protein expression in TNBC has been investigated, including gene expression analyses and large-scale genomic studies (7-10). However, to date, no clear biological differences have been identified between HER2-low and HER2-null TNBC tumors.

In 2021, Denkert et al. reported a pooled analysis of four prospective clinical trials where patients were treated with NAC, demonstrating that patients with HER2-low tumors had better prognoses than those with HER2-null tumors; this trend was also observed in the hormone receptor-negative subgroup (11). Since then, several similar studies have been published, and while some have reported better outcomes for HER2-low patients (12), others have found no significant prognostic difference between HER2-low and HER2-null tumors (13,14), and no consistent conclusion has been reached.

Thus, the differences between HER2-low and HER2-null statuses among TNBC have not been fully investigated, and it remains to be determined whether HER2-low breast cancer can be clearly defined as a prognostically distinct subtype. Therefore, we conducted an analysis focusing on HER2-low expression in patients with TNBC who received NAC, aiming to examine whether any biological differences exist between HER2-low and HER2-null tumors.

Patient selection. A total of 261 patients with TNBC who underwent curative surgery after NAC between January 2008 and December 2018 at four hospitals (Tokyo Medical University Hospital, Tokyo Medical University Hachioji Medical Center, Yokohama City University Hospital, and Yokohama City University Medical Center) were included in this study. Clinicopathological features of the patients are shown in Table I. The mean age was 54.9 years (range=26-79 years), and 76.6% were clinical stage I/II. As to chemotherapy regimens, 77% of patients received both anthracycline and taxane, while 23% received only one of these agents for various reasons. ICIs were not used during the study period.

This study was approved by the ethics committee of Tokyo Medical University (Approval Number: T2020-0184) and the institutional review boards of each institution. A waiver of written informed consent was granted on the basis of the study’s retrospective nature. Patients could see the research plan on the website of the hospitals and were offered the choice to opt out of the study at any time.

Pathological assessment. TNBC was defined as tumors with estrogen receptor and progesterone receptor expression both less than 1%, and negative for HER2. HER2 negativity was defined according to the 2018 ASCO/CAP HER2 testing guidelines as immunohistochemistry 0, 1+, or 2+ with negative in situ hybridization (15). pCR was defined as the absence of invasive cancer in both the breast and axillary lymph nodes (ypT0/isN0).

Statistical analysis. JMPpro18 statistical software (SAS Institute, Inc., Cary, NC, USA) was used for statistical analyses. For comparisons of mean values, unpaired t-tests were employed. Comparisons between the two groups in contingency tables were conducted employing Fisher’s test. For analysis of distant metastasis-free survival (DMFS) and overall survival (OS), a Cox proportional hazard model was applied. For the full-model analysis, we selected variables based on their known clinical significance, including age, tumor grade, HER2 protein expression, clinical stage, NAC regimen and pCR. Kaplan-Meier curves were also calculated, and the log-rank test was applied for comparisons of survival distributions between patient groups. A p-value <0.05 was considered to indicate a significant difference.

Comparison between HER2-null and HER2-low tumors. Among the patients included in this study, 77 patients (29.7%) obtained pCR. Clinicopathological factors were compared between two groups based on HER2 status (HER2-null vs. HER2-low; Table II). A significant difference was observed only in the Ki67 labeling index, which was significantly higher in the HER2-null group compared to the HER2-low group (p=0.004). Although there was no statistically significant difference in pCR rates, HER2-low tumors showed a slightly lower pCR rate compared to HER2-null tumors (26.0% vs. 34.5%). A further comparison of pCR rates according to HER2 expression is shown in Figure 1. When comparing the three groups (HER2 0, 1+, and 2+), a trend toward a higher pCR rate was observed in the HER2-null group.

Factors relating to patient outcomes. During the mean observation period of 77 months (range=7-195 months), 46 patients developed distant metastasis (17.6% of the 261 cases). Forty-two patients (16.1%) died from all causes. Multivariate analysis revealed that tumor grade, clinical stage, NAC regimen, and pCR were independently associated with DMFS (p=0.003, p<0.001, p=0.018, and p=0.009, respectively; Table III). In other words, patients with low-grade tumors, clinically advanced disease, those who received NAC with either anthracycline or taxane alone, or those with residual invasive disease in the breast and/or lymph nodes had significantly shorter DMFS. No statistically significant difference was observed between HER2-low and HER2-null tumors.

Regarding OS, only tumor grade, clinical stage, and pCR were identified as independent factors (p=0.008, p<0.001, and p=0.038, respectively; Table IV). Kaplan-Meier analysis confirmed that there was no significant difference in DMFS or OS between patients with HER2-low and HER2-null tumors (Figure 2), but patients with HER2-low tumors tended to have a better DMFS (p=0.401).

In this study, among patients with TNBC treated with NAC, no statistically significant differences in pCR rate or patient outcomes were observed between those with HER2-low and HER2-null tumors. However, those with HER2-low tumors showed a slightly lower pCR rate and tended to have slightly better DMFS compared to those with HER2-null tumors.

The relatively lower pCR rate observed in HER2-low tumors may be explained by a lower proportion of basal-like features compared to HER2-null tumors, which are more proliferative and sensitive to chemotherapy. Basal-like tumors have a distinct genetic profile among TNBC, characterized by the expression of biomarkers, such as CK5/6 and EGFR, as well as a high frequency of TP53 mutations (16,17). Furthermore, these tumors have been reported to be highly sensitive to chemotherapy (18). Therefore, the higher pCR rate observed in the HER2-null group may reflect the biological characteristics of basal-like tumors.

One of the major limitations of this study is the relatively small number of patients and its retrospective nature. Additionally, since the patients were collected from multiple institutions, there were some variations in the chemotherapy regimens used. Although we discussed the possibility that HER2-null tumors may represent basal-like tumors, we were unable to perform a detailed analysis of the biological differences between HER2-low and HER2-null tumors, including assessing markers, such as EGFR. Future studies are warranted to explore whether similar or distinct outcomes will be observed under ICI-based treatments.

In this study, no significant differences were observed in pCR rates or outcomes between the HER2-low and HER2-null tumor groups among patients with TNBC treated with NAC. However, the HER2-low tumor group showed lower proliferative activity and a trend toward lower pCR rates. Furthermore, DMFS tended to be slightly better in this group compared to the HER2-null tumor group. These findings suggest that HER2-null tumors may represent a biologically distinct subgroup, potentially enriched with basal-like tumors. Future studies are warranted to determine whether similar trends are observed in recent treatment regimens incorporating ICIs.

The Authors have no conflicts of interest to disclose in relation to this study.

MI, YH, RW, KA, AY, and KN collected the clinical data. MI and YH conducted the data analysis and statistics. MI and YH drafted the original manuscript and KY, HK, and TI substantively revised it. All Authors revised and approved the final version of the article.

The Authors sincerely appreciate Clear Science Pty Ltd for language editing.

No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.