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Introduction

Breast cancer is the most common site-specific cancer and the leading cause of cancer-related death in women aged 20 to 39 years (1). The disease is a current global health problem, and more than 1 million cases are newly diagnosed each year (2). It is estimated that 1 in 8 of women in the United States (12.4%) is affected by invasive breast cancer in their lifetime (3). Moreover, in 2018, approximately 64,000 woman were diagnosed with in situ breast cancer, and approximately 41 thousand of women in the US died from this disease (4). However, approximately 50% of breast cancer incidence globally and 60% of breast cancer mortality occur in middle- to low-income countries, including Thailand, where the age-standardized incidence of breast cancer was 28.5 per 100,000 from 2010 to 2012 (2,5). Currently, strategies for breast cancer diagnosis include imaging and pathological studies, and clinicopathological aspects such as tumor size, TNM stage, hormone receptor status, and molecular subtype are used for therapeutic planning and prognostication (6).

β2-microglobulin (β2-M) is a low-molecular-weight protein consisting of a single chain of 100 amino acids that is a part of the invariant light chain of the HLA antigen molecule (7,8). It is expressed on the membrane of almost all nucleated cells and is detectable in all body fluids as a shedding product of cell membranes (9,10). Ninety percent of β2-M is eliminated through glomerular filtration and is almost completely reabsorbed by the proximal convoluting tubules. At the clinical level, serum and urine β2-M concentrations are used to monitor glomerular and tubular nephropathies (11,12). The levels of serum and urine β2-M are also increased in patients with neoplastic diseases, including multiple myeloma, lymphoma and leukemia (13-16). Increased serum β2-M levels reflect increased cellular turnover rate and disease progression in some hematologic malignancies (17,18). For example, a β2-M value of less than 4 µg/ml was found to correlate with better survival in multiple myeloma (19). Increased β2-M serum levels in patients with breast cancer has been reported (20,21). An immunohistochemical (IHC) study reported that serum β2-microglobulin levels in patients with breast cancers were significantly higher than those in patients with benign breast tumors. In addition, the expression levels of β2-M protein in breast cancer tissue were found to be significantly different among patients with the 4 molecular subtypes (22,23). However, the clinical value of serum β2-M as a prognostic marker and predictor of survival needs further study (24).

The aim of this study was to evaluate the association between serum β2-M levels and the clinicopathological characteristics of breast cancer patients, especially the intrinsic subtypes and clinical stages. In addition, an in vitro study of the influence of β2-M on the cellular migration of a breast cancer cell line was conducted.

Materials and methods

Subjects and study protocols

The study design was a prospective cohort. Serum samples from a total of 200 female patients with histologically confirmed invasive breast cancer at Songklanagarind Hospital and adequate pathological data were collected from 2017 to 2019 after informed consent was obtained. The exclusion criteria included those with abnormal renal function, those who had previously received any form of treatment for breast cancer and those with other cancers. Pathologic stage and cancer subtype were identified according to the AJCC 8th Edition (25) and St. Gallen International Expert Consensus 2013(26), respectively. Metastatic work-up included abdominal ultrasound and chest computerized tomography. The metastatic status of all patients was confirmed by radiology and histopathological evidence. Treatment of breast cancer in our institute followed the Adult Cancer Treatment Guideline of Thai National Health Security Office (2018) with individual adjustment according to the functional status. Patients were appointed every 3 months during the period of active treatment, then every 6 months, thereafter. The Human Research Ethics Committee of the Faculty of Medicine, Prince of Songkla University, approved the study protocol (Reference no. 60-040-10-1).

Serum β2-M

Serum β2-M level was measured once at the time of breast cancer diagnosis. Blood was collected under aseptic precautions. Serum was separated and immediately analyzed by immunological agglutination with latex reaction enhancement assay using Immunoturbidimetric Assay kit (Roche/Hitachi). The ratio of reaction is 2:180:80 for sample:buffer:latex suspension. The concentration of β2-microglobulin was evaluated by measuring the agglutination reaction at 570 nm compared to the absorbance of standard β2-microglobulin.

IHC staining

The expression of ER, PR, HER-2 and Ki67 in tumor tissues was evaluated by IHC staining with the following primary antibodies: Anti-ER (Thermo Scientific, Inc., clone SP1, 1/250 dilution), anti-PR (Leica Biosystems, 1/2500 dilution), anti-Ki-67 antigen (Dako Glostrup, 1/250 dilution), and anti-HER2 (Ventana). The staining results were reported by a certified pathologist as the number of positive cells per 100 cancer cells. In cases with equivocal HER2 results, the specimens were further studied by HER-2 dual in situ hybridization (HER2-DISH).

HER2-DISH

HER2 and chromosome 17 probes were used for two-colour chromogenic in situ hybridization of formalin-fixed paraffin-embedded human breast cancer specimens following the VENTANA BenchMark XT automated slide staining protocol. The slides were evaluated under light microscopy for HER2 and chromosome 17 signals in at least 20 nuclei. Calculation of the HER2/chromosome 17 ratio was performed by dividing the total number of HER2 signals in the target area by the total number of chromosome 17 signals in the same area.

Cell migration assay

The cell migration assay was conducted in a 24-well plate with Transwell chambers (8-µm pore PET membrane; Falcon, Fisher Scientific). The low migratory cell line, MCF-7, obtained from American Type Culture Collection (ATCC; Catalog HTB-2), and routinely cultured on monolayers at ≤80% confluence in RPMI-1640 medium (Gibco) containing 10% FBS at 37˚C, 5% CO2 was used in this study. After starvation for 24 h, 2x105 cells in 100 µl 1% FBS RPMI-1640 medium with or without recombinant β2-microglobulin (Abcam) were added to the upper compartment of the chamber, whereas the lower chamber contained RPMI-1640 with 10% FBS. After 72 h of incubation at 37˚C, the cells on the upper surface were removed using a cotton swab. The membranes were fixed with 70% ethanol for 10 min at room temperature and stained with 0.1% crystal violet for 10 min. The number of migrated cells was quantified by counting cells in five different fields of view under a light microscope at a magnification of 200x. The data are presented as the mean ± SD.

Statistical analysis

The association between β2-M levels and clinicopathological factors was analyzed by using unpaired Student's t-test or one-way ANOVA with Tukey's post hoc test. As age at diagnosis was a major confounder, patients were subcategorized into 2 groups on the basis of age, those aged 25-55 years and those aged >55 years, for analysis of the association between metastatic status and β2-M level. For the Transwell migration study, one-way ANOVA followed by Turkey's post hoc test was used to determine statistical significance between groups. A receiver operating characteristic (ROC) curve was plotted using the sensitivity and specificity of each β2-M cut-off that predicted metastatic status. Survival analysis used log rank test and Kaplan-Meier survival plot with cancer related death used as a censor in overall survival (OS) analysis. Beginning date used operative date and survival data were as of December 2020. All data were analyzed with Statistical Package Stata 14.0 (Stata Corporation). A P-value of less than 0.05 was considered statistically significant.

Results

Patients and clinical data

A total of 200 patients were included in this study. The mean age at the time of diagnosis was 54 years (range 25-88 years). The most common histological type was invasive ductal carcinoma (188 cases; 94%), where the most frequent tumor grade was grade III (83 cases; 41.5%). The percentage of patients with positive lymphovascular invasion was 36.5% (73 cases). Lymph node involvement was most frequently seen in the N0 group (121 cases; 60.5%). T2 was the highest group among the T stages (108 cases; 54%). The percentage of patients with distant metastasis was 4% (8 cases). Regarding tumor molecular subtypes, luminal B was the most common tumor subtype, followed by luminal A (Table I). The example of HER-2 staining and HER-2-DISH was showed as Fig. S1.

Table I Serum β2-M levels in patients with breast cancer according to pathological parameters.

Table I

Serum β2-M levels in patients with breast cancer according to pathological parameters.

Variable	No. (%)	Average β2-M, µg/ml (range)	P-value
Total cases	200 (100.0)	1.83 (0.5-4.2)
Age, years			<0.01a
0-55	110 (55.0)	1.64 (0.5-4.0)
>55	90 (45.0)	2.05 (0.9-4.2)
Tumor side			0.70a
Right	101 (50.5)	1.84 (1.0-4.2)
Left	99 (49.5)	1.81 (0.5-4.0)
Histologic type			0.02a
Invasive ductal carcinoma	188 (94.0)	1.80 (0.5-4.2)
Lobular carcinoma and others	12 (6.0)	2.21 (1.9-2.5)
Tumor grade			0.41b
Grade-1	36 (18.0)	1.79 (1.0-3.8)
Grade-2	81 (40.5)	1.89 (1.1-4.0)
Grade-3	83 (41.5)	1.77 (0.5-4.2)
Lymphovascular invasion			0.54a
No	127 (63.5)	1.84 (0.9-4.2)
Yes	73 (36.5)	1.79 (0.5-4.0)
T-stage			0.17b
T1	75 (37.5)	1.79 (1-3.8)
T2	108 (54.0)	1.81 (0.5-4)
T3	9 (4.5)	2.26 (1.3-4.2)
T4	8 (4.0)	1.79 (1.3-2.3)
N-stage			0.72b
N0	121 (60.5)	1.85 (0.9-4.0)
N1	52 (26.0)	1.83 (0.5-4.2)
N2	18 (9.0)	1.71 (1.3-2.5)
N3	9 (4.5)	1.71 (1.2-2.1)
M-stage			<0.01a
M0	192 (96.0)	1.80 (0.5-4.0)
M1	8 (4.0)	2.40 (1.4-4.2)
Clinical stage			0.15b
Stage 1	58 (29.0)	1.83 (1.0-3.8)
Stage 2	104 (52.0)	1.81 (0.5-4.0)
Stage 3	30 (15.0)	1.70 (1.2-3.1)
Stage 4	8 (4.0)	2.40 (1.4-4.2)
ER status			0.75a
Negative	42 (21.0)	1.80 (0.9-4.0)
Positive	158 (79.0)	1.83 (0.5-4.2)
PR status			0.74a
Negative	68 (34.0)	1.84 (0.9-4.2)
Positive	132 (66.0)	1.81 (0.5-4.0)
HER2 status			0.71b
Negative	133 (66.5)	1.82 (0.5-4.0)
Equivocal	22 (11.0)	1.92 (1.3-3.3)
Positive	45 (22.5)	1.81 (0.9-4.2)
Intrinsic subtype			0.54b
Luminal A	78 (39.0)	1.77 (0.5-4.0)
Luminal B	88 (44.0)	1.89 (0.9-4.2)
HER-2	18 (9.0)	1.72 (0.9-2.6)
Triple negative	24 (12.0)	1.85 (1.2-4.0)

[i] ^aComparisons of β2-M between subgroups were performed using an unpaired Student's t-test.

[ii] ^bComparisons of β2-M among subgroups were performed using one-way ANOVA. β2-M, β-2 microglobulin; ER, estrogen receptor; PR, progesterone receptor.

β2-M levels were associated with metastatic status and survival rate in breast cancer patients

The serum β2-M levels in female breast cancer patients according to each pathological parameter are shown in Table I. Statistically significant differences in β2-M levels were found to be associated with age, histologic type, and metastatic status. As our data showed that serum β2-M levels were correlated with age, the level was re-analyzed with age stratification. The association between β2-M levels and metastatic status held true only in those aged >55 years (Table II).

Table II Comparing serum β2-M levels stratified by age group.

Table II

Comparing serum β2-M levels stratified by age group.

Age group	β2-M levels in no metastasis group, µg/ml	β2-M levels in metastasis group, µg/ml	P-valuea
25-55 years (N 106:4)	1.63	1.98	0.14
>55 years (N 86:4)	2.01	2.82	0.01

[i] ^aComparison was made using Student's t-test (unpaired; two-sided). N represents the number of cases in the non-metastasis group:number of cases in the metastasis group. β2-M, β-2 microglobulin.

To evaluate the correlation between serum β2-M levels and the prediction of metastatic status in breast cancers, a receiver operating curve (ROC) was constructed, as shown in Fig. 1. The area under the curve (AUC) was 0.78. If a serum β2-M level of 1.9 µg/ml was used as a cut-off, the sensitivity to diagnose the metastatic status was 87.5%, the specificity was 65.0%, and the diagnostic odds ratio was 2.47. Details of the diagnostic value of β2-M using different cut-off values are shown in Table SI. When the ROCs of β2-M performance for diagnosing metastasis were replotted by age subgroup, the AUC of the group of age 25-to 55-year-old group and the >55-year-old group were 0.75 (logistic regression P-value 0.22) and 0.82 (P-value 0.03), respectively. Median follow-up duration was 27.8 months and 24-month OS in all cases was 95.7%. Two-year overall survival (2-year OS) analysis was performed by cancer types, stages, and β2-M levels. The results showed that no significance of difference was found in 2-years OS when patients were sub-grouped by cancer types (log rank P-value=0.07) (Fig. 2A). However, when sub-grouping by stage of cancer, the significance of 2-year OS was found with the percentage of 96.4, 99.0, 88.2 and 75.0% for stages I-III and IV, respectively (log rank P-value <0.01) (Fig. 2B). Two-year OS in cases with serum β2-M levels lower than 1.9 µg/ml (98.3%) was also significantly higher than that of high serum β2-M (89.3%, log rank P-value <0.01) (Fig. 2C).

Figure 1 ROC curve of the sensitivity and 1-specificity of serum β-2 microglobulin levels in the prediction of metastatic disease in breast cancer. ROC, receiver operating characteristic.

Figure 2 Kaplan-Meier curves comparing survival probability. (A) Overall survival according to the intrinsic subtype. (B) Overall survival according to stage grouping. (C) overall survival according to serum β2-M levels. β2-M, β-2 microglobulin.

β2-M promoted migration ability of human breast cancer cell line

To study the influence of β2-M on the metastatic capability of breast cancer, the low migratory MCF-7 cell line was subjected to the Transwell cell migration assay. Cells were treated with human recombinant β2-M at different concentrations and incubated for 72 h. The results revealed that ectopic treatment with β2-M stimulated MCF-7 breast cancer cell migration, especially at 30 µg/ml, and that the number of migrated cells was decreased at lower concentrations (Fig. 3). These findings indicated that β2-M had a direct functional effect on MCF-7 cell movement.

Figure 3 Transwell migration assay of MCF-7 cells treated with β2-microglobulin at different concentrations. The upper panel shows a representative microscopic view (magnification, x200) of the migrating cells that reached the lower chamber. The lower panel shows the migrating cell numbers/field. The number in the 30 µg/ml group was significantly higher than those in the group treated with a lower concentration (15 µg/ml) and the no treatment control. *P<0.05.

Discussion

Human leukocyte antigen (HLA) consists of a heavy chain containing the alpha1, 2 and 3 domains and a light chain, which is a linked form of β2-M. β2-M can be dissociated from HLA molecules and found as a free form in extracellular fluid and can also be detected in the blood (27,28). Generally, the levels of β2-M is directly dependent on the kidney's function and cell turnover rate; however, some studies have found correlation between the serum level of this protein and several cancers (29). The association between female breast cancer and tissue expression β2-M has been observed in previous reports (22,23). Higher serum β2-M in breast cancer patients has been reported since 1977 by Transdale and colleagues (30). The mean serum β2-M in our cases was lower than that reported by Teasdale. Interlaboratory variation may explain this disparity. Our main findings of an association between high serum β2-M levels and metastatic status in breast cancer were consistent with recent studies which reported higher serum β2-M levels in metastatic breast cancer patients than in those with early-stage or locally advanced diseases (31). In addition, our study also provided the suggested cut-off value of β2-M at 1.9 µg/ml that might predict metastatic status and also demonstrated poorer survival probability in patients with high serum β2-M.

Roles of β2-M in the promotion of the epithelial to mesenchymal transition (EMT) in cancers, which is the key scenario in cancer metastasis have been reported (32). Overexpression of β2-M in cancers induces the invasion and migration ability of breast, lung, and renal cancer cell lines (33). In addition, β2-M induces bone and soft tissue metastasis in mice, and it can be used as a therapeutic target (29,33). This evidence supports our findings and recent studies reporting high serum levels of β2-M in cancer patients with distant metastasis (34). In accordance with our results, when poorly migrating cancer cells were exposed to high levels of recombinant β2-M, a higher migration ability was observed. This finding supports the role of β2-M in promoting cancer cell metastasis. However, the detailed mechanism of β2-M in cancer metastasis should be further evaluated. Our data suggested that serum β2-M might be used as a marker for advanced disease at the time of diagnosis or during post-treatment follow-up.

Another consideration is that our data showed that serum β2-M levels significantly varied with age, which may be related to the fact that β2-M excretion decreases with a reduction in glomerular filtration rate in the elderly. This same physiological process may explain the significant association between the serum β2-M level and metastasis in the group of patients aged >55 years but not in the younger patients. Intact renal function and rapid clearance of β2-M from the circulation results in less exposure of cancer cells to this migration-promoting substance. The limitations of our study were its cross-sectional design; in which no chronological data on alteration of β2-M level can be analyzed. However, our data was consistent with a previous study in that patients with metastatic breast cancer and high β2-M significantly had poorer survival (35).

In conclusion, serum β2-M levels were significantly higher in women with metastatic breast cancer than in those with cancer of less advanced stages. In addition, the high level was correlated with poorer survival outcome. Serum β2-M may be a non-invasive marker of metastatic status in breast cancer, and the cut-off level of 1.9-2.0 µg/ml might be used for metastatic prediction with a greater than 85% sensitivity.

Supplementary Material

Immunohistochemical and DISH analysis of HER-2 in breast cancer. (A) Immunohistochemical score 0. No staining on tumor cell membrane. (B) Immunohistochemical score 1. Faintly perceptible staining on >10% tumor cell membrane. (C) Immunohistochemical score 2. Moderate staining on >10% tumor cell membrane. (D) Immunohistochemical score 3. Strong staining on >10% tumor cell membrane. (E) Case with HER-2 amplification using DISH analysis. Pink color indicates the reference probe of Chr. 17 centromere, while brown color is the target probe for HER-2. Scale bars, 200 μm. DISH, dual color in situ hybridization.

Sensitivity, specificity and positive likelihood ratio of each β2-microglobulin cut-off value in predicting metastatic breast cancer.

Acknowledgements

The authors would like to thank Mr. David Patterson of the International Affairs Office, Faculty of Medicine, Prince of Songkla University (Hat Yai, Songkhla, Thailand) for manuscript proofreading and language editing.

Funding

This research was supported and funded by the Faculty of Medicine, Prince of Songkla University (grant no. REC 60-040-010-1) with postdoctoral fellowship program of the Faculty of Medicine, Prince of Songkla University.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

SM and SSan conceived the study. SJ, SM, SSam, KK, JS and SSan developed the methodology. SSan provide software. SM and SSan validated the data. SJ, SSan, JS and SM performed formal analysis. SJ, KK and JS performed investigations. SSam and KK provided resources. SJ, SSan and SM curated data. SJ and JS wrote the original draft. SSan and JS reviewed and edited the manuscript. SSan and SM visualized data. SM supervised the study. SM was the project administrator. SM acquired funding. The authenticity of all the raw data was confirmed by SSan and SM. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The board of The Human Research Ethics Committee of Faculty of Medicine, Prince of Songkla University approved the study protocol (reference no. 60-040-10-1; Hat Yai, Songkhla, Thailand). Written informed consent was obtained at the time of original sample collection.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References