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IMR Press / CEOG / Volume 49 / Issue 6 / DOI: 10.31083/j.ceog4906135
Open Access Original Research
Analysis of the Clinical Molecular Characteristics and Neoadjuvant Chemotherapy Response in Patients with Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer and Axillary Lymph Node Metastasis
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1 Department of General Surgery, Yantai Penglai People's Hospital, 265600 Yantai, Shandong, China
2 Department of Breast Center, The Affiliated Hospital of Qingdao University, 266000 Qingdao, Shandong, China
*Correspondence: wanghaibowhb91@126.com (Hai-Bo Wang)
These authors contributed equally.
Clin. Exp. Obstet. Gynecol. 2022 , 49(6), 135; https://doi.org/10.31083/j.ceog4906135
Submitted: 22 February 2022 | Revised: 21 March 2022 | Accepted: 28 March 2022 | Published: 7 June 2022
This is an open access article under the CC BY 4.0 license.
Abstract

Objective: This study aimed to investigate the clinical molecular characteristics in patients with human epidermal growth factor receptor 2 (HER2)-negative breast cancer and axillary lymph node metastasis and explored the related factors of the neoadjuvant chemotherapy (NAC) response. Methods: The data of 185 patients with HER2-negative breast cancer and axillary lymph node metastasis who were treated in the Department of Breast Center of the Affiliated Hospital of Qingdao University from July 2017 to July 2021 were retrospectively analyzed. The clinical features and the related factors for the responses of the primary tumor and axillary lymph node metastasis to NAC were analyzed. Statistical analysis was conducted using the SPSS 26.0 statistical software. Univariate analysis was conducted using the $\chi{}$${}^{2}$ test, and multivariate analysis was conducted using logistic regression analysis. Results: The differences in estrogen receptor (ER), progesterone receptor (PR), and Ki67 among the three HER2-negative subgroups (the immunohistochemistry (IHC)0 group, IHC1+ group, and IHC2+/in situ hybridization– group) were statistically significant (p $<$ 0.05). Univariate analysis revealed that the differences in the tumor stage, ER, PR, and Ki67 among the groups based on the response of the primary tumor to NAC were statistically significant (p $<$ 0.05), and the differences in ER, PR, and Ki67 among the groups based on the response of axillary lymph node metastasis to NAC were statistically significant (p $<$ 0.05). Multivariate analysis revealed that the difference in Ki67 among the groups based on the response of axillary lymph node metastasis to NAC was statistically significant (p $<$ 0.05). Conclusions: When the expression level of HER2-negative IHC increases, the positive rates of ER and PR increase. A smaller tumor, negative ER, negative PR, and a Ki67 level $>$30% indicate a good effect of NAC for primary tumors. Negative ER, negative PR, and a Ki67 level $>$30% indicate a good effect of NAC for axillary lymph node metastasis. Therefore, Ki67 may be an independent factor affecting the efficacy of NAC for axillary lymph node metastasis.

Keywords
HER2-negative
breast cancer
axillary lymph nodes
1. Introduction

Breast cancer is the most commonly occurring cancer among women worldwide. As the incidence continues to increase, this disease seriously endangers the physical and mental health of women [1].

The HER2 protein expression level is assessed by immunohistochemistry (IHC) and in situ hybridization (ISH) [7]. A HER2-negative result includes a low HER2 expression (IHC1+ or IHC2+ and fluorescence in situ hybridization-negative) and a zero HER2 expression (IHC0). Antibody-coupled drugs provide new therapeutic options for patients with breast cancer, including those with a low expression of HER2. The latest study published in The Lancet [8] revealed that the hormone receptor-positive rate, pCR rate, disease-free survival rate, and overall survival rate were different between low HER2 expression tumors and zero HER2 expression tumors. Low HER2 expression tumors have unique biological characteristics, different therapeutic effects, and survival results, especially for drug-resistant and hormone receptor-negative tumors.

In the present study, the investigators studied the factors related to NAC for HER2-negative breast cancer, primary breast cancer, and axillary lymph node double metastasis breast cancer. Furthermore, HER2-negative cancers were divided into three subgroups to study their clinical molecular characteristics; this is helpful for subtype refinement and precise treatment of HER2-negative breast cancer. For patients with poor efficacy of NAC, preliminary screening can be performed based on clinical molecular characteristics, and patients with low HER2 expressions can be treated with antibody–drug conjugate (ADC) drugs.

2. Information and Methods
2.1 Clinical Data Acquisition

The data of patients with breast cancer who were treated in the Department of Breast Center of the Affiliated Hospital of Qingdao University from July 2017 to July 2021 were retrospectively analyzed. Neoadjuvant chemotherapy and surgery were performed in the Department of Breast Center.

2.2 Inclusion Criteria

The inclusion criteria were as follows: if the primary tumors and axillary lymph node tumors were diagnosed as invasive carcinoma by a core needle biopsy; patients who were female; if the immunohistochemical information included estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67; if there was no presence of distant metastasis; patients who had not received malignant tumor-related treatment before the treatment administered in this study; if the first-line regimen of NAC was anthracycline combined with paclitaxel or anthracycline sequential paclitaxel; patients who had undergone a modified radical mastectomy.

2.3 Exclusion Criteria

The exclusion criteria were as follows: if the primary tumor had been diagnosed as a carcinoma in situ or a specific type of cancer; women who were pregnant or lactating; if the immunohistochemical information was uncertain or insufficient; patients with distant metastasis; patients who had received malignant tumor-related treatment before the treatment administered in this study; if there had been a change of NAC regimen; if a mastectomy had not been performed or an axillary lymph node dissection had not been completed.

2.4 Data Acquisition and Database Establishment

Patients who were HER2-negative were divided into three groups: an IHC0, IHC1+, and IHC2+/ISH– group. The NAC responses of the primary tumors were divided into the G1–G3 and G4–G5 groups according to the Miller–Payne grades. The treatment responses of the axillary lymph node tumors were divided into the CR of axillary lymph nodes (apCR) group and the non-CR of axillary lymph nodes (non-apCR) group according to the remission rates. Age, body mass index (BMI), menarche age, menstrual status, clinical tumor stage (cT), ER, PR, HER2, Ki67, and pCR were collected.

2.5 Statistical Analysis

Data were statistically analyzed using the SPSS 26.0 statistical software (IBM Corp, Armonk, NY, USA). Clinical molecular characteristics and related factors of the treatment response of the primary tumor and axillary lymph node metastasis were compared among the HER2-negative breast cancer subgroups using the $\chi{}$${}^{2}$ test or Fisher’s exact probability method. Univariate analysis was conducted using the $\chi{}$${}^{2}$ test or Fisher’s exact probability method to screen out significant variables, which were then included in the multivariate logistic analysis. A p value of $<$0.05 was considered statistically significant.

3. Results
3.1 Basic Data of the Patients

In this study, 185 patients with HER2-negative breast cancer and axillary lymph node metastasis were enrolled. In 23 patients, primary tumors and axillary lymph node metastasis achieved CR. In 13 patients, only primary tumors achieved CR, and in 24 patients, only axillary lymph node metastasis achieved CR (Table 1).

Table 1.Patient demographic and clinical characteristics at baseline.
 Characteristic Total population, n (%) Age $<$40 29 (15.68%) 40–60 117 (63.24%) $>$60 39 (21.08%) BMI (Kg/m${}^{2}$) $\leq$25 89 (48.11%) $>$25 96 (51.89%) Menarche age $<$14 42 (22.70%) 14–16 112 (60.54%) $>$16 31 (16.76%) Menstrual status Premenopausal 91 (49.19%) Postmenopausal 94 (50.81%) cT T1 24 (12.97%) T2 88 (47.57%) T3 40 (21.62%) T4 33 (17.84%) ER status (biopsy) Negative 41 (22.16%) Low-Positive (1–10%) 11 (5.95%) High-Positive ($>$10%) 133 (71.89%) PR status (biopsy)* Negative 64 (34.59%) Low-Positive (1–20%) 43 (23.24%) High-Positive ($>$20%) 78 (42.16%) HER2 (biopsy) IHC0 66 (35.68%) IHC1+ 87 (47.03%) IHC2+/ISH– 32 (17.30%) Ki67 expression (biopsy) $<$15% 12 (6.49%) 15–30% 67 (36.22%) $>$30% 106 (57.30%) PCR aPCR 24 (40.00%) bPCR 13 (21.67%) PCR 23 (38.33%) Note: *According to CSCO guidelines, PR20% serves as the threshold for Luminal A and Luminal B.
3.2 Clinical Molecular Characteristics in the Different Subgroups of HER2-Negative Breast Cancer

There were 66 cases of IHC0, 87 cases of IHC1+, and 32 cases of IHC2+/ISH–. There were no significant differences in age, BMI, menarche age, menstrual status, cT, primary tumor treatment response, or axillary lymph node metastasis treatment response among the HER2-negative subgroups. The differences in ER, PR, and Ki67 among the three HER2-negative subgroups were statistically significant (p $<$ 0.05, Table 2).

Table 2.Association between clinicopathological factors and HER2-negative subtypes.
 Characteristics n IHC0 IHC1+ IHC2+/ISH– $\chi$${}^{2}$ p age $<$40 29 11 12 6 40–60 117 40 59 18 $>$60 39 15 16 8 1.655 0.799 BMI (Kg/m${}^{2}$) $\leq$25 89 29 43 17 $>$25 96 37 44 15 0.843 0.656 Menarche age $<$14 42 19 16 7 14–16 112 39 55 18 $>$16 31 8 16 7 3.507 0.477 Menstrual status Premenopause 91 35 46 10 Postmenopausa 94 31 41 22 4.982 0.083 cT T1 24 12 7 5 T2 88 27 49 12 T3 40 12 21 7 T4 33 15 10 8 10.265 0.114 ER Negative 41 22 15 4 Positive 144 44 72 28 7.728 0.021 PR Negative 63 31 25 7 Positive 122 35 62 25 8.112 0.017 Ki67 $<$15% 19 5 5 9 15–30% 60 18 36 6 $>$30% 106 43 46 17 17.774 0.001 Response of primary tumor G1–G3 129 44 61 24 G4–G5 56 22 26 8 0.721 0.697 Response of axillary lymph nodes apCR 47 18 21 8 Non-apCR 138 48 66 24 0.198 0.906
3.3 Analysis of Related Factors for the Response of Primary Tumors to NAC

Among the patients, there were 129 cases with a Miller–Payne grade of G1–G3 and 56 cases with a Miller–Payne grade of G4–G5.

3.3.1 Univariate Analysis

There were no significant differences in age, BMI, menarche age, menstrual status, cT, HER2, or ER among the groups based on the response of the primary tumor to NAC. The differences in ER, PR, and Ki67 among the groups based on the response of axillary lymph node metastasis to NAC were statistically significant (p $<$ 0.05, Table 3).

Table 3.Association between clinicopathological factors and primary tumors response.
 Characteristics n G1–G3 G4–G5 $\chi^{2}$ p age $<$40 30 18 12 40–60 116 83 33 $>$60 39 28 11 1.607 0.448 BMI (Kg/m${}^{2}$) $\leq$25 89 56 33 $>$25 96 73 23 3.767 0.052 Menarche age $<$14 42 29 13 14–16 112 74 38 $>$16 31 26 5 3.656 0.161 Menstrual status Premenopause 76 55 21 Postmenopausa 109 74 35 0.426 0.514 cT T1 24 12 12 T2 88 61 27 T3 40 28 12 T4 33 28 5 8.008 0.046 HER2 IHC0 66 44 22 IHC1+ 87 61 26 IHC2+/ISH– 32 24 8 0.721 0.697 ER Negative 40 22 18 Positive 145 107 38 5.246 0.022 PR Negative 62 33 29 Positive 123 96 27 12.034 0.001 Ki67 $\leq$30% 79 66 13 $>$30% 106 63 43 12.466 0.000 Response of axillary lymph nodes apCR 47 18 29 Non-apCR 138 111 27 29.491 0.000
3.3.2 Multivariate Analysis

Estrogen receptor, PR, Ki67, and the response of axillary lymph node metastasis to NAC were included in the binary logistic regression for multivariate analysis. The results revealed that there was a significant difference in the response of axillary lymph node metastasis to NAC (p $<$ 0.05, Table 4).

Table 4.Multivariate logistic regression analysis for primary tumors response.
 Clinicopathological factors B SE Wald p OR (95% CI) ER –0.45 0.524 0.737 0.391 0.638 (0.228–1.781) PR 0.802 0.454 3.127 0.077 2.230 (0.917–5.425) Ki67 –0.731 0.422 2.996 0.083 0.482 (0.211–1.102) Response of axillary lymph nodes 1.58 0.409 14.946 0.000 4.854 (2.179–10.814)
3.4 Analysis of Related Factors for the Response of Axillary Lymph Node Metastasis to NAC

There were 47 cases of apCR and 138 cases of non-apCR.

3.4.1 Univariate Analysis

There were no significant differences in age, BMI, menarche age, menstrual status, cT, and HER2 among the groups based on the response of axillary lymph node metastasis to NAC, and the differences in ER, PR, and Ki67 among the groups based on the response of the primary tumor to NAC were statistically significant (p $<$ 0.05, Table 5).

Table 5.Association between clinicopathological factors and axilla response.
 Characteristics n apCR Non-apCR $\chi^{2}$ p age $<$40 29 9 20 40–60 117 26 91 $>$60 39 12 27 1.703 0.427 BMI(Kg/m${}^{2}$) $\leq$25 89 23 66 $>$25 96 24 72 0.017 0.895 Menarche age $<$14 42 7 35 14–16 112 35 77 $>$16 31 5 26 5.119 0.077 Menstrual status Premenopause 91 25 66 Postmenopausa 94 22 72 0.404 0.525 cT T1 24 8 16 T2 88 26 62 T3 40 7 33 T4 33 6 27 3.820 0.282 HER2 IHC0 66 18 48 IHC1+ 87 21 66 IHC2+/ISH– 32 8 24 0.198 0.906 ER Negative 41 20 21 Positive 144 27 117 15.187 0.000 PR Negative 63 27 36 Positive 122 20 102 15.353 0.000 Ki67 $\leq$30% 79 7 72 $>$30% 106 40 66 19.915 0.000 Response of primary tumor G1–G3 129 18 111 G4–G5 56 29 27 29.491 0.000
3.4.2 Multivariate Analysis

Estrogen receptor, PR, Ki67, and the response of the primary tumor to NAC were included in the binary logistic regression for multivariate analysis. The results revealed that there were significant differences in Ki67 between the responses of axillary lymph node metastasis to NAC and the responses of primary tumors to NAC (p $<$ 0.05). These results indicate that Ki67 (OR = 3.571, 95% CI: 1.386–9.201, p = 0.008) may be an independent factor affecting the response of axillary lymph node metastasis to NAC (Table 6).

Table 6.Multivariate logistic regression analysis for axilla response.
 Clinicopathological factors B SE Wald p OR (95% CI) ER –0.655 0.492 1.773 0.183 0.520 (0.198–1.362) PR –0.446 0.463 0.930 0.375 0.640 (0.258–1.585) Ki67 1.273 0.483 6.950 0.008 3.571 (1.386–9.201) Response of primary tumor 1.551 0.396 15.324 0.000 4.715 (2.169–10.250)

In this study, ER, PR, and Ki67 were expressed differently in the three HER2-negative subgroups, and the negative-to-positive ratio of ER and PR decreased gradually in the IHC0, IHC1+, and IHC2+/ISH– subgroups. The negative-to-positive ratio of ER was 50.00%, 20.83%, and 14.29%, respectively, and the negative-to-positive ratio of PR was 88.57%, 40.32%, and 28.00%, respectively. In HER2-negative breast cancer, when the expression level of IHC increases, the positive rates of ER and PR increase.

Univariate analysis revealed that the efficacy of NAC for primary tumors might be related to the tumor stage, ER, PR, Ki67, and the response of axillary lymph node metastasis to NAC. The efficacy of NAC for axillary lymph node metastasis may be related to ER, PR, Ki67, and the response of primary tumors to NAC. Multivariate analysis revealed that Ki67 might be an independent factor affecting the efficacy of NAC for axillary lymph node metastasis. A smaller tumor, negative ER, negative PR, and a Ki67 level $>$30% indicate a good effect of NAC on the primary tumor. Negative ER, negative PR, and a Ki67 level $>$30% indicate a good effect of NAC for axillary lymph node metastasis. Therefore, Ki67 may be an independent factor affecting the efficacy of NAC for axillary lymph node metastasis.

4. Discussion

The number of patients with breast cancer who receive neoadjuvant therapy is increasing [9]. Compared with adjuvant chemotherapy, NAC can dynamically monitor the drug sensitivity of tumors during chemotherapy, facilitate timely adjustment of drug dosage, and ensure the best curative effect of chemotherapy [10, 11]. In addition, NAC can reduce the clinical stage, increase the surgical resection and breast preservation rates, and greatly improve the quality of life and prognosis of patients. The five-year survival rate also significantly improves for patients who achieve pCR after NAC [12].

Breast cancer therapy enters a new level with ADC intervention. This treatment produces exciting results not only in patients with HER2-positive breast cancer but also in patients with low HER2 expression breast cancer [13, 14, 15, 16]. In this study, gene expression analysis revealed that a low expression of HER2 exists in luminal and non-luminal types of breast cancer, but ER is usually positive, especially in the luminal B type [17]. According to immunohistochemical expression levels, HER2-negative breast cancer can be subdivided into the IHC0, IHC1+, and IHC2+/ISH– groups, while low HER2 expression groups include IHC1+ and IHC2+/ISH– levels. We discovered differences in the clinical molecular characteristics among the three subgroups that showed that the conditions of a positive ER, positive PR, a Ki67 level $<$15%, and IHC2+/ISH– expression increased, which suggested that an IHC2+/ISH– expression is more common in the luminal A type. Luminal A is known to be insensitive to chemotherapy, and ADC agents are expected to provide a more feasible and effective treatment for these patients.

A previous study revealed that compared with zero HER2 expression, a low expression of HER2 was usually related to a higher histological grade and proliferation rate, and the prognosis was poorer [18, 19, 20, 21]. Recent data has revealed that the effective treatment rate of patients with HER2+ breast cancer was higher than that of patients with HER1+ breast cancer. Gentile et al. [22] established that there were no significant differences in age, histological grade, tumor size, lymph node status, and chemotherapy regimen between an effective and ineffective group. In unscreened invasive breast cancer, determining mass size under an ultrasound was a strong prognostic factor [23, 24]. In a study of women receiving NAC, determining tumor size under an ultrasound was not associated with metastasis-free survival, although there is evidence claiming that small tumors are more likely to achieve CR [25]. The Ki67 protein affects cell cycles and DNA synthesis, reflects the proliferation of tumor cells, and is also associated with the development and prognosis of breast cancer [26].

In this study, a smaller tumor, negative ER, negative PR, and a Ki67 level $>$30% indicated a good effect of NAC for primary tumors. Negative ER, negative PR, and a Ki67 level $>$30% indicated a good effect of NAC for axillary lymph node metastasis. Therefore, Ki67 may be an independent factor in the response of axillary lymph node metastasis. Based on existing data and experimental results, experts suggest that HER2 can be subdivided into the positive HER2, negative HER2 (zero expression of HER2), and low HER2 expression types to formulate different treatment regimens. Therefore, about 55% of breast cancers will be classified as a low expression of HER2. HER2 low expression breast cancer may be heterogeneous in the microenvironment of tumor-infiltrating lymphocyte enrichment, which may be related to antibody-dependent cytotoxicity [18]. However, there are different types of low HER2 expression heterogeneity, and whether it will lead to different degrees of treatment response remains to be determined. The dichotomous definitions of positive HER2 and negative HER2 are currently undergoing a series of changes. By identifying the low HER2 expression types and studying ADC drugs, HER2-negative breast cancer may be subdivided into low expression and zero expression of HER2 groups to formulate different treatment options.

5. Conclusions

HER2-negative breast cancer cannot be treated with targeted therapy, and the PCR rate of NAC is low, especially for hormone receptor-positive breast cancer. It is expected that early surgical or ADC intervention can help to achieve long-term benefits by screening patients with poor efficacy and poor prognosis through clinical analysis. However, this will require further study using a larger sample size.

Author Contributions

Conception and design of the research—YZ, JPM. Acquisition of data—JPM, JHZ, TW, YM. Analysis and interpretation of the data—JPM, JZ. Statistical analysis—JPM. Obtaining financing—YZ. Writing of the manuscript—JPM, SF. Critical revision of the manuscript for intellectual content—HBW. All authors read and approved the final draft.

Ethics Approval and Consent to Participate

This study was conducted with approval from the Ethics Committee of Yantai Penglai People’s Hospital (202013). This study was conducted in accordance with the declaration of Helsinki. Written informed consent was obtained from all participants.

Acknowledgment

We would like to acknowledge the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study.

Funding

Scientific research project of Qingdao University Medical Group (No. YLJT20202020).

Conflict of Interest

The authors declare no conflict of interest.

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