Bioinformatics combined with clinical data to analyze clinical characteristics and prognosis in patients with HER2 low expression breast cancer

Introduction

Breast cancer (BC) is a highly heterogeneous disease at histological and genetic levels, and the clinicopathological and molecular genetic characteristics of different types of BC are quite different (1). Perou first proposed in 2000 molecular typing based on gene expression profiles (2). Through the subsequent continuous supplementation and improvement, in 2011, the St. Gallen International Breast Cancer Conference panel combined fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). According to the different combinations of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67, BC can be divided into four subtypes: luminal A, luminal B, HER2 overexpression, and triple negative type. There are obvious differences in the treatment options and prognosis of different subtypes of BC (3,4). The HER2 gene has become an important indicator in the molecular typing, treatment guidance, and prognostic analysis of BC.

The past 20 years have seen great improvements in the prognosis of HER2-positive (HER2⁺) BC patients, and the quality of life and survival of patients have markedly improved. This has been confirmed by many large clinical studies, and molecular targeted therapy has become an indispensable treatment for HER2⁺ BC (5,6). Based on the 2018 edition of American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) HER2 detection guidelines, a study by Wolff et al. and Onsum et al. showed that different degrees of HER2 protein expression exist in some HER2-negative (HER2⁻) BC patients (7,8). At present, these patients are still classified as the luminal and triple negative breast cancer (TNBC) type, and their treatment is also limited to the treatments methods of the luminal and TNBC types, which does not constitute precision treatment of BC. In recent years, with the development of anti-HER2 drugs, the therapeutic efficacy of HER2⁺ BC has significantly improved, and some HER2 low expressing BC patients can also benefit from them, which has triggered a wave of research on HER2 low expressing BC, and several guidelines have also proposed the concept of HER2 low expression (9-12). However, at present, there are few research reports on this type of BC, there is still a lack of strong evidence separately listed as a new BC subtype, and there is no specific target for molecular targeted treatment, resulting in uneven clinical treatment effect and prognosis.

Unlike HER2⁺ BC, wherein the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-47 test showed that most tumors with low or moderate HER2 expression and no amplification could currently not be targeted and inactivated by conventional anti-HER2 drugs (13). However, the results of recent clinical trials show that novel antibody-drug conjugates (ADCs), including trastuzumab-deruxtecan (T-DXd) and trastuzumab-duocarmazine (SYD985) (14-17), are clinically active, which is not only in classical HER2⁺ BC, but also in tumors with low-to-moderate HER2 expression levels (18). The development of anti-HER2 ADC provides new therapeutic options for BC patients, including those with HER2 low expression.

The Cancer Genome Atlas (TCGA) as the largest cancer gene information database, TCGA not only includes gene expression data, miRNA expression data, copy number variation, DNA methylation, etc., but also contains rich and standardized clinical data. Treatment strategies for various subtypes of BC are different based on their distinct molecular characteristics. Gan et al. analyzed the relevant data in Gene Expression Omnibus (GEO) database were analyzed by bioinformatics method, and key differential expression genes (DEGs) between ER-positive/HER2⁻ BC and ER-negative/HER2⁻ BC were found, and three differential genes affecting the prognosis of the two were further analyzed, which provided a therapeutic direction for the treatment of HER2⁻ BC (19).

Historically, HER2⁻ BC has not been used as a target for conventional anti-HER2 therapy. However, in recent years, two anti-HER2 ADCs have also shown good anti-tumor activity in BC patients with HER2 low expression, thus providing a new treatment option for patients with HER2 low expression BC, and this new specific type of BC has become a research hotspot in the past 2 years. So far, there is still a lack of reliable research on HER2 low expression BC in China.

HER2 low expression as a specific type of BC is currently less studied, in order to describe this new BC subtype, this study first downloaded BC data through TCGA database, analyzed and compared the difference between HER2 low expression group and HER2 zero expression group and its correlation with ER and PR expression, and then analyzed the clinical sample data to verify the conclusion of TCGA database, so as to provide theoretical and experimental support for the new diagnosis and treatment strategy of this future category of BC, and improve its survival and efficacy. We present the following article in accordance with the REMARK reporting checklist (available at https://gs.amegroups.com/article/view/10.21037/gs-22-747/rc).

Methods

Research process

In this study, the breast cancer (BRCA) raw data were first downloaded from TCGA database to analyze the difference between HER2 low expression and HER2 zero expression in terms of clinicopathological characteristics, and its correlation with ER and PR expression. Then, HER2 expression was detected in clinical samples by IHC and FISH, cases were divided into the HER2 low expression group and HER2 zero expression groups, and relevant case data were collected to verify the TCGA data.

Raw data

The TCGA-BRCA dataset downloaded from the TCGA database contained 345 BC samples with complete data, including 57 HER2 zero expression samples and 288 HER2 low expression samples. Clinical samples were obtained from 405 confirmed HER2⁻ BC patients admitted to the Affiliated Hospital of Youjiang Medical University for Nationalities (YJMU) from January 2017 to December 2021, including 284 with HER2 low expression and 121 with HER2 zero expression. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by institutional board of Youjiang Medical University for Nationalities (No. 2020020301). Informed consent was taken from all the patients.

Definition of HER2 low expression and HER2 zero expression

The IHC results of 1+ or 2+ and negative FISH results were defined as HER2 low expression, and negative IHC results were defined as HER2 zero expression.

Identification of the differentially expressed genes (DEGs) between HER2 low expression samples and HER2 zero expression samples

Of the 345 HER2⁻ BC patients, 57 patients had HER2 zero expression and 288 patients had HER2 low expression. Comparing patients with HER2 zero expression and HER2 low expression, the DEGs were obtained using the “limma” package in the R software (R Foundation for Statistical Computing, Vienna, Austria) with a threshold set to |log₂fold change (log₂FC)| >0 and false discovery rate (FDR) <0.05. The expression differences of ER and PR in both species were analyzed.

Statistical analysis

R software was used for database data analysis in this study, using the limma package for differential analysis between HER2 zero expression and HER2 low expression. The software SPSS 25.0 (IBM Corp., Armonk, NY, USA) was used to analyze clinical samples, and Kaplan-Meier method was used for survival analysis. Chi-square test was used to compare the rates of the two groups, non-parametric test was used for comparison between groups, and Spearman analysis was used for correlation analysis. A P value <0.05 was considered statistically different.

Results

Clinicopathological characteristics of BRCA patients with HER2 low expression and HER2 zero expression in TCGA database

The TCGA-BRCA database contained 57 patients with HER2 zero expression and 288 patients with HER2 low expression. Compared with the HER2 zero expression, ER and PR were higher (P=0.02) and PR was more positive (P=0.006) in HER2 low expression patients. However, there was no significant difference in tumor diameter, lymph node metastasis, and distant metastasis between the initial treatment groups (P>0.05) (Table 1).

Clinicopathological features of the HER2 low expression versus HER2 zero expression of BC in clinical samples

Clinicopathological data were collected from 405 patients with confirmed HER2⁻ BC admitted to YJMU from January 2017 to December 2021, including 284 patients with HER2 low expression and 121 patients with HER2 zero expression. Compared with the HER2 zero expression group, The ER positive rate was higher (P<0.0001), as was the PR positive rate (P=0.006). However, the age, menstrual status, tumor diameter, lymph node metastasis, distant metastasis at initial diagnosis, histological grade, and Ki-67 expression intensity were not significantly different between the two groups (P>0.05) (Table 2).

The DEGs between HER2 low expression and HER2 zero expression

Compared with HER2 zero expressed patients, a total of 133 DEGs were identified in HER2 low expressed patients, among which 22 were upregulated and 111 were downregulated (Figure 1A,1B).

Figure 1 The DEGs between HER2 zero expression and HER2 low expression. (A) A heatmap of the DEGs in BC patients. (B) A volcano plot of the DEGs in BC patients. HER2, human epidermal growth factor receptor 2; FC, fold change; DEGs, differentially expressed genes; BC, breast cancer.

The expression of the estrogen receptor 1 (ESR1) and progesterone receptor (PGR) in HER2 low expression and HER2 zero expression of patients

Expression of ESR1 and PGR was higher in patients with HER2 low expression compared to HER2 zero expression patients (Figure 2A,2B). HER2 expression in patients with HER2 low expression was positively correlated with the expression of ESR1 and PGR (Figure 3A,3B).

Figure 2 The expression levels of the ESR1 and PGR in HER2 zero expression and HER2 low expression patients. (A) ESR1. (B) PGR. HER2, human epidermal growth factor receptor 2; ESR1, estrogen receptor 1; PGR, progesterone receptor.

Figure 3 Correlation between the ERBB2 (HER2) expression level and the expression level of ESR1 (ER), and PGR (PR). (A) ESR1. (B) PGR. ERBB2 (HER2), human epidermal growth factor receptor 2; ESR1, estrogen receptor 1; PGR, progesterone receptor; HER2, human epidermal growth factor receptor 2; ER, estrogen receptor; PR, progesterone receptor.

Comparison of HER2 low expression in prognosis with HER2 zero expression in BC

The Kaplan-Meier survival analysis showed no significant difference in disease-free survival (DFS) between HER2 zero expression and HER2 low expression groups in TCGA dataset (P=0.892) and YJMU dataset (P=0.420) (Figure 4A,4B). Compared to the HER2 zero expression group, the HER2 low expression group had a lower rate of recurrence or metastasis at 3 years (P=0.018) (Table 3). In the subgroup analysis, when the hormone receptor (HR)-positive group was found, the 3-year recurrence or metastasis rate was lower than that in the HER2 zero-expression group (P=0.020) (Table 4).

Figure 4 Kaplan-Meier analysis of the DFS in the HER2 zero expression group and the HER2 low expression groups. (A) TCGA dataset; (B) YJMU dataset. DFS, disease-free survival; HER2, human epidermal growth factor receptor 2; TCGA, The Cancer Genome Atlas; YJMU, the Affiliated Hospital of Youjiang Medical University for Nationalities.

Discussion

According to the intensity of HER2 protein expression, BC was previously divided into HER2⁺ and HER2⁻ treatment strategies, whereby HER2 low expression was treated as HER2⁻, and could not benefit from anti-HER2-targeted therapy (20,21). Currently, HER2⁺ BC is defined as the overexpression of HER2 protein as detected by IHC judged as 3+ and/or FISH judged as positive (22,23). In 70–85% of the HER2⁻ BC, there are still low-to-moderate HER2 expression (IHC 1+/IHC 2+/FISH−) tumors known as HER2 low expression tumors (24). Previous experience of HER2⁻ BC indicated that it cannot be a target for conventional anti-HER2 therapy. However, in recent years, two ADCs targeting against HER2 such as T-DXd and SYD985 have also shown good anti-tumor activity in BC patients with HER2 low expression, thus providing new treatment options for HER2 low expression BC patients (25,26). This new specific type of BC has become a research hotspot in the past 2 years.

Based on the special pathological characteristics of the HER2 low expression that are different from those of HER2⁺ and HER2⁻ BC, this study analyzed the patients with HER2 low expression from the database and clinical samples, in order to find its particularity and provide a theoretical basis and experimental support for clinical treatment and prognostic analysis.

In this study, a total of 57 HER2 zero expression and 288 HER2 low expression samples were extracted from the TCGA database. We first analyzed the DEGs in the two groups and obtained 133 differential genes: 22 up-regulated expression genes and 111 down-regulated expression genes compared with the HER2 zero expression group, indicating that the pattern of HER2 low expression and HER2 zero expression were different. In addition, from the analysis of ER and PR expression level, both the positive expression rate of protein level and the molecular expression level of messenger RNA (mRNA) showed that the low expression group of HER2 was higher than that of the HER2 zero expression group. All the above results indicate that patients with HER2 low expression have completely different pathological characteristics from the patients with HER2 zero expression. The pooled analysis of individual patient data from four prospective neoadjuvant clinical trials showed that HER2 low expression was more positive for HR expression compared with HER2 zero expression. Zhang et al. showed that there were significantly more HR-positive patients in the HER2 low expression group than in the HER2 zero expression group (26). These findings are highly consistent with this study and indicate that HER2 zero expression and HER2 low expression BC should be included in molecular typing.

On further analysis, the gene expression level of HER2 was positively correlated with the expression of ER and PR. To further verify the above results, we included a total of 405 female BC patients from the clinical samples, with a high ER and PR positive rate in the HER2 low expression group compared with the HER2 zero expression group (P<0.0001). However, in our study, there were no significant differences in age, menstrual status, tumor diameter, lymph node metastasis, distant metastasis at initial diagnosis, histological grade, and Ki-67 expression intensity between the two groups (P>0.05). Research shows HER2 low expression is dynamic in BC and may be enriched in the advanced-stage setting, but there is no prognostic significance was demonstrated for HER2 low expression (27). Compared with the HER2 zero expression group, the recurrence or metastasis rate at 3 years was significantly reduced in the HER2 low expression group (P=0.018). In the subgroup analysis, it was found that the recurrence or metastasis rate at 3 years in the HR-positive group was significantly lower than that in the HER2 zero expression group (P=0.020), while there was no statistical difference in the recurrence or metastasis rate between the two groups in the overall population. Further Kaplan-Meier survival analysis showed that there was no significant difference in DFS between the HER2 zero expression group and the HER2 low expression group in the overall population (P=0.420). There was also no difference in 3-year DFS rates between the two groups (P=0.250). The results of this study are consistent with numerous reports at from China and internationally (28-30). However, when performing the prognostic analysis, the different HER2 expression intensity of the 345 BC patients in the TCGA cohort was not significantly different from the DFS, which is consistent with the DFS results of our clinical samples, and is also consistent with the findings of other centers in (30-32). However, in the group with HER2 low expression (P=0.018); in subgroup analysis, the rate with HER2 low expression was significantly lower than in the HER2 zero expression group (P=0.020). Currently, many studies have shown that HER2 low expression has a longer DFS compared with HER2 zero expression BC patients (33,34). Denkert et al. showed that low HER2 expression was associated with high 3-year DFS (P=0.0084) and 3-year overall survival (OS) compared with HER2 zero expression (P=0.0016) (34). For patients with HR-negative tumors, HER2 low expression showed a high 3-year DFS (P=0.0076) and 3-year OS (P=0.016) compared with zero HER2 expression; however, the 3-year DFS rate and the 3-year OS rate were not significantly different among the 2 patients with HR-positive tumors. Further studies found that compared with HER2 zero-expression tumors, the pCR rate was lower, and in patients with HR-negative tumors, the pCR rate was similar, while in patients with HR-positive tumors, the pathological complete response rate of HER2 low-expression tumors was significantly reduced compared with HER2 zero-expression tumors (35). Similarly, the study of 1,433 HER2⁻ BC initiated by Li et al. and Tan et al. found significantly longer survival in HER2 low expressing tumors than HER2 zero expressing tumors in the overall population and HR-positive subgroups, but showed no significant difference in the HR-negative subgroup (35,36). The results of a based on an ABCCG cohort study were nearly identical to the findings of Li et al. (35). Mutai et al. found that for patients at high risk of recurrence, the low HER2 expression had a better long-term prognosis compared with the HER2 zero expression (29). There are also other studies that show neither DFS and OS differences between HER2 zero expression and HER2 low expression nor HER2⁻ BC, which do not support low HER2 expression as a biologically distinct BC subtype or the effect of HER2 low expression on the survival of BC patients (29-31).

At present, the results of all studies in China and internationally showed that the HR of the HER2 low expression group was higher than the HER2 zero expression group in both the protein expression levels and the mRNA expression levels. Moreover, the gene expression level of HR was positively correlated with the HER2 gene expression level, which suggests that patients with low HER2 expressing BC have unique biology. The HER2 low expression and the HER2 zero expression of BC did not show a significant difference in the survival prognosis, which also shows the current lack of clinical diagnosis and treatment of this type of BC. These types of BC include molecular level and differential gene expression. At the same time, more accurate and intelligent pathology technology is also needed to accurately distinguish this type of BC, to enable scientific and precise individualized treatment of HER2, and to endow it with a better long-term prognosis.

Conclusions

In conclusion, this study clarified that the HER2 low expression in BC has different clinicopathological characteristics compared with the HER2 zero expression, and the investigators need to continue more dimensional research in HER2 low expression in BC patients to achieve better long-term prognosis.

Acknowledgments

Funding: This work was supported by the Guangxi Natural Science Foundation (Nos. 2020GXNSFAA259056, 2018GXNSFAA281038); Baise Scientific Research and Technology Development Program (No. encyclopedia 20220914); Self-funded project of Baise Scientific Research and Technology Development Plan in 2022 (No. Baiword 20220915); High-Level Talents Research Projects General Projects of the Affiliated Hospital of Youjiang Medical University for Nationalities (No. 202011719); First Batch of High-Level Talent Scientific Research Projects of the Affiliated Hospital of Youjiang Medical University for Nationalities in 2019 (No. R20196307); Research Project of Youjiang Medical University for Nationalities (No. 2019002).

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://gs.amegroups.com/article/view/10.21037/gs-22-747/rc

Data Sharing Statement: Available at https://gs.amegroups.com/article/view/10.21037/gs-22-747/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://gs.amegroups.com/article/view/10.21037/gs-22-747/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by institutional board of Youjiang Medical University for Nationalities (No. 2020020301). Informed consent was taken from all the patients.

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

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(English Language Editor: J. Jones)