In total, 50 women who were diagnosed with MBC and treated at the Department of Thyroid and Breast Surgery, Affiliated Hospital of Zunyi Medical University (Guizhou, China) between February 2010 and February 2021, were retrospectively included. During this period, a total of 3,081 patients were diagnosed with breast cancer, and MBC accounted for 1.59%. The main inclusion criteria were as follows: Female, without distant metastasis at first diagnosis and confirmed to be MBC by the Pathology Department of The Affiliated Hospital of Zunyi Medical University. The main exclusion criteria were as follows: Male, bilateral cancer, presence of distant metastasis and unavailability of tissue samples. A total of 49 patients were included in this study according to the aforementioned criteria, and 1 patient was excluded due to the inability to obtain tissue samples. All available clinicopathological data, including age, menstrual status, tumor size, lymph node status, TNM stage, immunohistochemistry (IHC) results and treatment were collected from the medical records. The patients received all therapeutic procedures, such as surgery, adjuvant CT, irradiation and hormonotherapy at the same institution (Table I).

The time to follow-up was from the date of surgery to the date of recent follow-up. Patient follow-up was accomplished by specialized staff at the Department of Thyroid and Breast Surgery, Affiliated Hospital of Zunyi Medical University, and routine correspondence and telephone calls were used for follow-up. The follow-ups were performed every 3 months during the first 2 years, every 6 months during the next 3 years, then once a year thereafter. Overall survival (OS) time was calculated from the date of surgery to the occurrence death of any cause. Disease-free survival (DFS) time was estimated from the date of surgery until the date of first proven recurrence, including local/regional recurrence and distant metastasis at any site. The last follow-up was conducted in April 2021. The current study was approved by the Ethical Committee of The Affiliated Hospital of Zunyi Medical University. All procedures were in accordance with the 1964 Declaration of Helsinki and its later amendments. Written informed consent was provided by all the patients, and all tissue samples used were from paraffin embedded tissues following surgery. The tumor tissue was fixed with 10% neutral buffered formalin at room temperature overnight, and the 4-µm thick tissue sections were used for pathological evaluation.

H&E-stained slides of the MBCs were reviewed according to the 2012 World Health Organization classification criteria (16). The histological sections were stained with hematoxylin for 8–10 min and eosin for 4–5 sec at room temperature, then the stained sections were observed under a light microscope (magnifications ×40 and ×100). PMBCs were defined as having a mucinous component of >90% and MMBC was defined with a 51–90% mucinous component. In addition, hypocellular MBC (type A) and hypercellular MBC (type B) were also determined based on cell cluster density (17).

ER, PR, HER-2 status and the Ki-67 index were evaluated using IHC. Briefly, the 4-µm thick tissue sections were incubated with the immunohistochemical antigen repair buffer (cat. no. MVS-0099; Beijing Strong Biotechnologies, Inc.) for 20 min after dewaxing in xylene for 60 min and rehydrated in a descending alcohol series (100, 95 and 75%) at room temperature. Subsequently, the tissue sections were blocked using an endogenous biotin blocking kit (cat. no. BLK-0002; Beijing Strong Biotechnologies, Inc.) for 10 min at room temperature. After washing with PBS, the tissue sections were incubated for 32 min at 42°C with primary antibodies targeted against ER (cat. no. kit-0012; clone SP1; 1:100; rabbit monoclonal), PR (cat. no. kit-0013; clone SP2; 1:100; rabbit monoclonal), HER2 (cat. no. Kit-0043; clone MXR001; 1:100; rabbit monoclonal) and Ki-67 (cat. no. RMA-0731; clone MXR002; 1:100; rabbit monoclonal) (all from Beijing Strong Biotechnologies, Inc.). After washing with PBS, the tissue sections were processed with a MaxvisionTM HRP kit (cat. no. kit-5004; Beijing Strong Biotechnologies, Inc.). The IHC results were judged by experienced pathologists using a light microscope (magnification, ×40 and ×100), and the ER and PR were regarded as positive if >1% of nuclei were stained (18). With respect to Ki-67, a range of 500–1,000 cells were counted to calculate the percentage of positive tumor cell nuclei, including hot spot areas (19). The molecular subtype was classified according to the 2013 St. Gallen expert panel consensus (20). All histological and IHC tumor slides were evaluated independently by two pathologists.

HER2 status was considered to be positive if >10% of the tumor cells showed a score of 3+ from IHC or showed a >2.2-fold increase in FISH using a HER2 DNA Probe kit (cat. no. 2J01-30; Abbott Molecular Inc.) (21). Briefly, after the samples were deparaffinized, dehydrated and air-dried, the tissue sections were handled with pre-treatment solution at 80°C for 30 min. Then, the sections were immersed in protease solution at 37°C for 34 min, followed by immersion in wash buffer (70, 80 and 100% ethanol). Subsequently, the tissue sections were incubated with the probe mixture [10 µl HER2 probe (226 kb; 10 ng/µl) and 10 µl CEP17 probe (9 kb; 20 ng/µl)] at 74°C for 5 min, then the cover slip was sealed with Fixogum rubber cement (cat. no. 12101ES62; Marabu GmbH and Co. KG) for 10 min at room temperature, and the samples were subsequently incubated overnight at 37°C. Next, the samples were washed with post-hybridization wash buffer at room temperature for 15 min. After air-drying, 10 µl DAPI (cat. no. 30-804840; Abbott Molecular Inc.) was added to the target area and a cover glass was added and the samples were incubated at −20°C for 10 min. After the slides were stored in the dark and left at room temperature, the FISH results were judged by experienced pathologists using a fluorescence microscope (magnification, ×40 and ×100).

The Oncotype DX 21-gene test was performed by AmoyDx Diagnostics Co., Ltd. Briefly, the H&E-stained slides were reviewed by pathologists to ensure that the paraffin section contained sufficient tumor tissue. RNA was then extracted from the unstained breast tumor formalin fixed paraffin-embedded (FFPE) sections using a RNeasy FFPE RNA kit (cat. no. 172348; AmoyDx Diagnostics Co., Ltd), and the concentration was measured after verifying the absence of DNA contamination. Gene-specific RT was performed at 65°C for 5 min and 37°C for 60 min using the PrimeScript RT Master Mix kit (Takara Biotechnology, Co., Ltd.). Subsequently, standardized quantitative PCR was performed using Premix Ex Taq™ (Takara Bio, Inc.) in 384-well plates and an Applied Biosystems Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the following thermocycling conditions were used: Initial denaturation at 95°C for 10 min, 95°C for 20 sec and 60°C for 45 sec (for 40 cycles). The 16 genes examined comprised of five proliferation-related genes [Ki-67, aurora kinase A (AURKA), baculoviral IAP repeat containing 5 (BIRC5), cyclin B1 (CCNB1) and MYB proto-oncogene like 2 (MYBL2)], two metastasis-related genes [MMP11 and cathepsin V (CTSV)], two HER2-related genes [growth factor receptor bound protein 7 (GRB7) and HER2], four hormone-related genes [ER, PR, BCL2 and signal peptide CUB domain and EGF like domain containing 2 (SCUBE2)] and three independent genes [glutathione S-transferase mu 1 (GSTM1), BAG cochaperone 1 (BAG1) and CD68], which were normalized according to five reference genes (ACTB, GAPDH, RPLP0, GUSB and TFRC). Therefore, 16 cancer-related genes in 21 genes can be used to predict the outcome of patients. The expression of the genes was confirmed in triplicate, and the relative gene expression was calculated using the 2^−∆∆Cq method (22), and the RS was calculated based on the Oncotype DX formula (10). According to the RS results, patients were categorized into low-risk (RS <18), intermediate-risk (RS ≥18-30) and high-risk (RS ≥30) groups (23). For further analysis, the individual gene expression of the 16-cancer genes was measured, and the distribution of the 16-cancer gene expression in PMBC and MMBC cases was analyzed.

The clinicopathological characteristics were presented as patient number and percentage and the other data was expressed as the mean ± standard deviation and range. The χ² test or Fisher's exact test were used to evaluate associations between PMBC and MMBC, while the Kruskal-Wallis test was used to compare quantitative characteristics. Logistic regression was used in multivariate analyses to identify risk factors impacting lymph node metastasis. The Kaplan-Meier estimation (log-rank test) was used to assess DFS and OS rate, and the Cox proportional hazard model was used to analyze the prognostic factors of patients with MBC. The Mann-Whitney test was used to assess the distribution of the 21-gene RS in the different subgroups, and to compare the expression levels of the 16 cancer genes between subgroups. P<0.05 was considered to indicate a statistically significant difference. SPSS version 22.0 software (IBM Corp.) was used for all the statistical analyses.

In total, 49 cases diagnosed as MBC (38 PMBCs and 11 MMBCs) were included in this analysis and the pathological changes of various typical MBCs are shown in Fig. 1. The median age at diagnosis was 52.3 ± 12.8 years (range, 33–87 years), and 44.9% of these patients were postmenopausal. The median tumor size was 3.2 ± 1.8 cm (range, 1.0–8.5 cm) at diagnosis, and 29.2% of cases had axillary lymph node involvement. According to IHC and FISH results, 45 (91.8%) and 38 (77.6%) patients with MBC were ER and PR positive, respectively. In 5 (10.2%) of the patients with MBC, HER2 positivity was detected, while 34.7% of all patients had ≥20% Ki-67 expression. For the molecular subtype, 49.0% (n=24) were classified as luminal A, 42.8% (n=21) as luminal B, 4.1% (n=2) as HER2-rich and 4.1% (n=2) as triple negative. The detailed clinicopathological characteristics of the patients are shown in Table II.

The mean age at diagnosis in patients with PMBC and MMBC was 51.5 ± 13.4 years (range, 33–87 years) and 54.9 ± 10.8 years (range, 33–78 years), respectively (P=0.25), and the mean tumor size in PMBCs and MMBCs was 3.19 ± 1.8 cm (range, 1.2–8.5 cm) and 3.17 ± 1.6 cm (range, 1.0–5.0 cm), respectively (P=0.914). The data showed no significant differences between PMBCs and MMBCs with respect to TNM stage (P=0.261), molecular subtype (P=0.17), status of ER (P=0.562), status of PR (P=0.398) and Ki-67 expression (P=0.395). However, a significantly higher incidence rate of axillary lymph node involvement was observed in MMBCs comparison with that in PMBCs (72.7 vs. 16.2%, respectively; P=0.001). The clinicopathological characteristics of the PMBCs and MMBCs are detailed in Table III. Similarly, the results of multivariate analysis demonstrated that the only high-risk factor of lymph node metastasis in patients with MBC was the pathological subtype (P=0.018; Table IV). Furthermore, the status of HER2 had a marginal P-value (P=0.068) in the two groups, and a higher incidence rate was observed in MMBCs compared with that in PMBCs (27.3 vs. 5.3%).

A total of 98.0% of the patients with MBC in the present study underwent radical mastectomies (1 patient refused surgery), and the first-line treatment selections following surgery in the MBC cases with different subtypes are presented in Fig. 2A. Overall, 8.2% (n=4), 36.7% (n=18) and 51% (n=25) of enrolled patients received CT, endocrine therapy (ET) and CT followed by ET as first-line treatment according to the molecular subtypes, respectively. The detailed adjuvant treatment of the MBC cases with various molecular subtypes are shown in Table I. Of all the patients with HER2 expression amplification, only 1 patient (20%) received trastuzumab therapy. In the PMBC and MMBC cases, the proportion of those receiving CT was 55.3 and 72.7%, respectively, and there was no statistical significance (P=0.102; data not shown).

The mean follow-up time for patients with MBC was 65.6 months (range, 2–125 months), and 2 patients were lost during this time. As shown in Fig. 2B and C, the 5-year DFS and 5-year OS rates for MBC was 97 and 100%, respectively, and this result was not statistically significant between PMBCs and MMBCs (log-rank test; P=0.457).

During the study period, distant metastases were found in 5 patients with high TNM stage (3 cases with stage III and 2 cases with stage II), and 2 of these patients died from lung metastases (both HER2 expression positive). In addition, 1 patient with PMBC with no recurrence died of a cardiovascular accident. The causes of treatment failure in MBCs cases are presented in Table V. In addition, Cox multivariate analysis did not identify any statistically significant factors associated with the prognosis of patients with MBC (Table VI).

In the present study, 29 of the 42 enrolled ER-positive and HER2-negative MBC cases underwent Oncotype DX 21-gene testing (the sample quality of 13 cases did not meet the test) and the results were evaluable, which included 21 PMBCs and 8 MMBCs. According to the criteria of 21-gene test RS stratification, 51.7% patients (15/29) were in the low-risk group (RS <18) with a mean RS of 10.5 ± 5.6, 44.8% patients (13/29) were in the intermediate-risk group (RS ≥18-30) with a mean RS of 22.3±5.2, and 3.5% patients (1/29) were in the high-risk group (RS ≥30) (RS, 35.7). The proportions of low-, intermediate- and high-risk RS were 42.9, 52.3 and 4.8%, respectively, among PMBCs, and 50.0, 50.0 and 0% in the MMBCs (P=0.91; Fig. 3A). The mean 21-gene RS in the PMBC and MMBC groups was 18.0 and 13.0, respectively (P=0.151; Fig. 3B). Notably, based on the traditional RS treatment recommendation, 37.9% of patients with MBC in the present study could avoid CT (Fig. S1).

The individual gene expression of the 16 cancer genes from the 21-gene test between the PMBC and MMBC groups was analyzed. The histograms of the distribution of cancer gene expression in the different histological-type subgroups are presented in Fig. 3C. In general, the expression levels of the genes from the proliferation and HER2 groups did not differ significantly between the PMBC and MMBC cases. In the metastasis group, the expression level of CTSV (P=0.005) was significantly higher in the PMBC group compared with that in the MMBC group. In the ER group, the expression level of PR (P=0.018) was significantly higher in the PMBC group compared with that in the MMBC group, and the expression level of ESR1 had a marginal P-value (P=0.053). Furthermore, in the independent group, the expression level of CD68 was higher in the PMBC group (P=0.003), while the expression levels of GSTM1 and BAG1 did not differ significantly between groups. The detailed expression of the 16 cancer genes between the PMBC and MMBC groups are shown in Table VII.