Natural killer cells are associated with prognosis in triple-negative breast cancer patients

doi:10.21203/rs.3.rs-1961278/v1

Objective:

Immune cells play an important role in anti-tumor activity. We aimed to investigate the expression of CD4+ and CD8+ T cells, natural kill (NK) cells, and interleukin-27 (IL-27) in breast cancer tissues, and to explore their relationship with clinical characteristics and patients’ prognoses.

Methods:

Immunohistochemistry was used to detect the expression levels of CD4+ and CD8+ T cells, NK cells, and IL-27 in a total of 198 breast cancer patients.

Results:

It was found that the number of positive NK cells was highest in patients with triple negative breast cancer (TNBC). In the Luminal A breast cancer group: the higher the histological grade, the higher the number of positive cases of CD4+ T cells; the higher the clinical stage, the higher the percentage of positive CD8+ T cells. In the Luminal B breast cancer group: the higher the histological grade, the higher the number of positive cases of CD8+ T cells; the higher the clinical stage, the higher the number of positive cases of CD8+ T cells. In the grouping of HER-2 breast cancer, patients with lymph node metastasis had higher CD8+ T cell positive cases; larger tumor volume had higher IL-27 positive cases. In TNBC grouping: the larger the tumor volume, the higher the positive ratio of CD8+ T cells; the higher the number of NK cell positive cases in patients with lymph node metastasis; the higher the number of IL-27 positive cases in patients with larger tumor volume. The survival curve showed that the OS rate of NK cell-negative patients was significantly higher than that of NK cell-positive patients.

Conclusion:

This study shows that infiltrating CD4+ and CD8+ T cells and NK cells in breast cancer tissue are associated with poor patient prognosis, and NK cell-positive TNBC patients with poor prognosis.

Breast cancer

Molecular subtypes

Immune cells

Immunotherapy

Breast cancer is the most common malignant tumor among women, ranking first among diagnosed cancers. Various comprehensive treatment methods are currently used, such as surgery, radiotherapy, chemotherapy, endocrine and immune therapies, and targeted drugs [1,2]. As a result, the overall prognosis for breast cancer is better than that of other cancers. However, the long-term survival rate for specific breast cancer types and advanced diseases can still be improved [3]. Therefore, new treatments would benefit more patients, reducing the cancer burden.

Molecular typing has identified four primary breast cancer subtypes: luminal A, luminal B, human epidermal growth factor receptor-2 (HER-2) overexpression, and triple-negative breast cancer (TNBC) [4]. Therefore, breast cancer prognoses and treatment responses vary widely due to high molecular heterogeneity. However, tumor immunology studies have identified associations between immune cells in the breast cancer microenvironment and prognoses for different pathological types, indicating that immune cells may be involved in immune tolerance and escape mechanisms. Thus, these mechanisms may provide new breast cancer treatment strategies [5,6].

T cells have a crucial role in the immune system. There are two primary subgroups of mature T cells: cluster of differentiation CD4+ and CD8+ [7,8]. These two subgroups regulate each other and the overall cellular immune response. Natural killer (NK) cells are another important innate immune cell that secrete cytokines and chemokines, inducing anti-tumor effects through immune clearance and surveillance [9]. Moreover, interleukin (IL)-27 is a heterodimeric cytokine that specifically affects CD4+ and CD8+ T cells, increasing interferon-gamma (IFN-γ) production in T cells thus promoting naive T cell proliferation. IL-27 also cooperates with IL-12 to regulate IFN-γ production by naive T cells [10,11] and enhances IFN-γ expression in NK cells to generate stronger T cells and exert an anti-tumor effect, inhibiting tumor progression [12]. Finally, IL-27 recruits NK cells to the surrounding tumor tissue by regulating chemokine expression and eliminating tumor cells [13].

Many studies have reported correlations between immune cells and breast cancer prognoses. However, the immune cell patterns per molecular subtype remain unclear. Therefore, this study used immunohistochemical methods to investigate CD4+ and CD8+ T cells, NK cells, and IL-27 expression in breast cancer tissue of different molecular types to clarify the immune cell infiltration patterns. In addition, by analyzing differences in the expression levels of these indicators, we aimed to elucidate relationships between their expression, clinical characteristics, and patient survival to provide a theoretical basis for novel clinical immunotherapies and individualized treatments.

Patients and samples

The study was approved by the ethical board of the People’s Hospital of Xinjiang Uygur Autonomous Region. We retrospectively analyzed the surgical specimens of tumor patients with primary breast cancer who underwent surgery in the Xinjiang Uygur Autonomous Region People’s Hospital, Xinjiang, China, from February 2012 to June 2016. Breast cancer was diagnosed with clinical pathology. The median age of the patients was 54 years with the range from 39 to 80 years. All patients received standard clinical treatment according to the National Comprehensive Cancer Network (NCCN) Guidelines. All of the clinicopathologic data were available. No patients had received neoadjuvant chemotherapy, and patients with bilateral breast cancer or non-epithelial breast malignancies and those lost to follow-up were excluded to eliminate compounding effects. We collected data on estrogen receptor (ER), progesterone receptor (PR), HER2, Ki-67 status by reviewing immunohistochemically (IHC) stained sections [14]. (ASCO-CAP Guideline Recommendations for Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer June 1, 2010 - See more at: http://www.instituteforquality. org/ practice-guidelines# sthash. fTzpaGZL. dpuf).

For each of the invasive tumors, diameter, tumor grade, ER status, PR status and HER2 status were listed. For all patients, the age, time of diagnosis, postoperative pathology (histological grade, tumor size, metastatic lymph node) clinical stage and follow-up were recorded.

Immunohistochemistry

A total of 4-micrometer-thick serial sections 195 paraffin-embedded breast cancer and breast fibroma tissue samples were obtained. All specimens were obtained from the pathology department of our hospital and analyzed using the IHC method. The sections were deparaffinized for 20 min in xylene, rehydrated in an alcohol gradient and washed with distilled water, then immersed in 3% hydrogen peroxide in methanol for 10 min to inhibit endogenous peroxidase. Antigen retrieval was carried out by placing the slides in citrate buffer (pH 6.0) for 2.5 min using a pressure cooker. The following antibodies were used: monoclonal mouse anti-AR (dilution, 1:200; ZSGB, ZM-0437, CHN) and polyclonal Rabbit anti-HDAC6 (dilution, 1:200; Abcam, ab1440, UK). The slides were blocked with 10% normal goat serum at 20 to 25°C for 30 min. Slides were then incubated with primary antibodies overnight at 4°C. In the negative control, no primary antibody was added. After washing with phosphate belanced solution, the slides were detected with second antibody and avidin-biotin complexing reagent. Afterwards, the slides were then incubated with horseradish peroxidase at room temperature for 50 min. Finally, the slides were counterstained with hematoxylin, removed in xylene, and mounted. Microscopic examination was used, and image acquisition and analysis were carried out. The nucleus of hematoxylin staining was blue, and the positive expression of DAB was brownish yellow.

Judging standard

Five visual fields were observed at a high magnification (400×). To determine the proportion and the staining intensity of cells with CD4, CD8, NK, and IL-27, staining was interpreted as positive if staining was present in more than 10% of the tumor cells, and expression was scored based on staining intensity (0, negative; 1, mild; 2, moderate; 3, strong) and the proportion of positive cells (1, 25%; 2, ≥25% and <50%; 3, ≥50% and <75%; 4, ≥75%) [15]. The IHC scores were calculated by multiplying the intensity and the proportion of positive cells, as previously described.

Statistical analysis

All statistical analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). The count data were analyzed using the Chi-square test. The two-tailed Fisher’s exact test was conducted where Chi-square test was not applicable. OS was analyzed using the Kaplan–Meier method, and its’ statistical significance was determined using the log-rank test. P < 0.05 was considered statistically significant.

Immunohistochemical Staining

After immunohistochemical staining, the nuclei were blue. Furthermore, NK cells and cells expressing IL-27 and C-X-C motif chemokine ligand 10 (i.e., CXCL10) were diffuse yellow and brownish-yellow granules. CD4+ and CD8+ T cells and NK cells were primarily distributed around mammary duct cells and scattered in the mammary glands; cells expressing IL-27 were diffusely distributed in the cytoplasm. NK cells were abundant in TNBC tissues (Figure 1).

CD4+ and CD8+ T cells, NK cells, and IL-27 expression

There were no differences in CD4+T cells, CD8+T cells and IL-27 among breast cancer groups. Regarding NK cells, we identified 30 NK cell-positive and 20 NK cell-negative luminal A cases, 29 positive and 21 negative luminal B cases, 33 positive and 17 negative HER-2 cases, and 36 positive and 9 negative TNBC cases (four-group comparison: χ² = 10.714, P = 0.013, Figure 2)

CD4+ and CD8+ T cells, NK cells, and IL-27 expression and clinical characteristics

We evaluated CD4+ and CD8+ T cells, NK cells, and IL-27 expression in each breast cancer subtype based on age, histological grade, clinical stage, tumor size, and lymph node metastasis (Tables 1–4).

For the luminal A subtype, the CD4+ T cell positive ratio significantly increased as the histological grade increased (χ² = 6.032, P = 0.049). Furthermore, the CD8+ T cells positive ratio significantly increased as the clinical stage increased (χ² = 7.669, P = 0.022).

For the luminal B subtype, the CD8+ T cell positive ratio significantly increased as the histological grade increased (χ² = 6.237, P = 0.041). Furthermore, the CD8+ T cell positive ratio significantly increased as the clinical stage increased (χ² = 9.644, P = 0.006).

For the HER-2 subtype, significantly more CD8+ T cells were identified in patients with lymph node metastasis than in patients without metastasis (χ² = 4.748, P = 0.029). Also, the IL-27 positive ratio significantly increased as the tumor volume increased (χ² = 4.115, P = 0.042).

Finally, for TNBC, the CD8+ T cell positive ratio significantly increased as the tumor volume increased (χ² = 4.946, P = 0.026). Also, significantly more patients with lymph node metastasis were NK cell-positive than patients without metastasis (P = 0.017). Furthermore, the IL-27 positive ratio significantly increased as the tumor volume increased (P = 0.016).

Prognosis

Kaplan–Meier survival curves demonstrated that CD4+ and CD8+ T cells, NK cells, and IL-27 expression correlated with overall survival (OS; Figure 3).

In each subgroup, we compared the OS curves of patients positive and negative for CD4+ T cells, CD8+ T cells, NK cells, and IL-27 expression. For the luminal A subtype, the log-rank (Mantel–Cox) values and P-values were 0.661 and 0.416 for CD4+ T cells, 0.475 and 0.491 for CD8+ T cells, 1.579 and 0.209 for NK cells, and 0.988 and 0.320 for IL-27 expression, respectively. For the luminal B subtype, the log-rank (Mantel–Cox) values and P-values were 0.123 and 0.726 for CD4+ T cells, 0.496 and 0.481 for CD8+ T cells, 0.914 and 0.339 for NK cells, and 0.110 and 0.740 for IL-27 expression, respectively. For the HER-2 subtype, the log-rank (Mantel–Cox) values and P-values were 0.001 and 0.976 for CD4+ T cells, 0.014 and 0.907 CD8+ T cells, 0.376 and 0.540 for NK cells, and 0.021 and 0.886 for IL-27 expression, respectively. Finally, for TNBC, the log-rank (Mantel–Cox) values and P-values were 2.276 and 0.131 for CD4+ T cells, 0.138 and 0.710 for CD8+ T cells, and 0.173 and 0.677 for IL-27 expression, respectively. Notably, the log-rank (Mantel–Cox) value was 3.981, and the P-value was 0.047 for NK cells, indicating that NK cell-positive patients had worse OS than NK-cell negative patients.

Years of research have resulted in a breakthrough in breast cancer immunotherapy. In 2019, the Phase III IMpassion130 study results were presented at the European Society of Medical Oncology annual meeting; these results suggested that breast cancer has officially entered an era of immunotherapy [16]. Immunotherapy mainly includes immune checkpoint inhibitors, cellular immunotherapy, cytokine therapy, tumor vaccines, and oncolytic viruses [17]. In addition, tumor immunotherapy aims to improve the immune state of the tumor microenvironment by stimulating or regulating the immune system, breaking tumor immunosuppression, and enhancing the killing effect on tumor cells [18]. Compared with traditional treatment methods, immunotherapy has a broad anti-cancer effect, strong specificity, good tolerance, non-toxic drug accumulation, and other noticeable therapeutic effects that considerably improve prognosis [18,19].

Our study found that TNBC tissue had significantly more NK cell infiltration than tissue from other subtypes. Furthermore, we identified a positive correlation between the CD4+ T cell positive ratio and the histological grade in the luminal A subtype and the CD8+ T cell positive ratio and the clinical stage in the luminal B subtype. Moreover, in the HER-2 and TNBC subtypes, the number of positive CD8+ T cells and IL-27 expression increased with tumor volume, and HER-2 positive breast cancer patients with lymph node metastasis had more positive CD8+ T cells than those without metastasis. Also, in the TNBC group, more patients with lymph node metastasis were positive for NK cells than those without metastasis. Finally, we found that TNBC NK cell-positive patients have a worse prognosis than NK-cell negative patients, consistent with our previous study [20]. These results emphasize that molecular subtypes of breast cancer have different immune cell infiltration patterns.

T cells are crucial to the adaptive immune response. Thus, harnessing the therapeutic potential of T cells could benefit breast cancer patients who respond poorly to conventional treatment methods, such as chemoradiotherapy [21]. Therefore, developing T-cell-based immunotherapy for advanced-stage breast cancer is worthwhile. Hirofumi et al. [22] performed immunohistochemical staining of CD4+ and CD8+ T cells in 164 TNBC tissues, finding a correlation between CD4+ and CD8+ T cells and the histological grade. Furthermore, survival analyses have shown that T cells with high CD4+ and CD8+ expression are good prognostic indicators in TNBC.

Moreover, Wen-Hui et al. [23] determined the percentage of CD4+ T cells in the peripheral blood of breast cancer patients by flow cytometry, reporting significantly lower amounts of CD4+ T cells in these patients than in the healthy controls. Additionally, the percentage of CD4+ and CD8+ T cells was significantly lower in stage II/III breast cancer patients than in stage I patients, and the immune function of breast cancer patients was significantly suppressed compared to that of healthy controls. Dora et al. [24] used immune-related genes to mark T cells in breast cancer tissues of different molecular types, finding that CD8+ T cell infiltration was the highest in basal-like and HER-2 type tissues, but other molecular types of breast cancer had higher degrees of infiltration. Furthermore, T cells with different functions and subpopulations are distributed in tumor tissue， suggesting that immunogenicity differences among breast cancers may indicate different tumor immunotherapy responses; this hypothesis provides a theoretical basis for combined immunotherapy based on the molecular subtype. Another study demonstrated that many T lymphocytes infiltrated TNBC tissues [25], and cancer tissues highly express programmed death-ligand 1 (PD-L1). Therefore, anti-PD-1/PD-L1 antibodies are essential for TNBC treatment. Now, the National Comprehensive Cancer Network guidelines include immunotherapy, changing the landscape of advanced breast cancer treatments.

NK cells are part of the innate immune system, and their potential role in cancer immunotherapy is currently under investigation. For example, Wenjing et al. [26] immunohistochemically detected NK cell infiltration in TNBC tissue, reporting that low-level NK cell infiltration was associated with poor OS. Furthermore, Somayeh et al. [27] assessed infiltrating NK cells in 32 breast cancer patients before treatment by flow cytometry, finding that the percentage of NK cells in the breast tumor tissue positively correlated with tumor size; the larger the tumor diameter, the higher the proportion of NK cells and the proportion of activated NK cells in patients without lymph node metastasis. Therefore, understanding the dynamic changes of NK cells may be an important reference for the immune function status of breast cancer patients, thus the treatment effects and prognosis.

IL-27 is an important cytokine connecting innate and adaptive immunity. It is primarily produced by activated antigen-presenting cells and acts on T cells, NK cells, and other immune cells, thus regulating anti-tumor immunity through various mechanisms. A clinical meta-analysis [28] found that the IL-27 gene rs153109 polymorphism increases tumor susceptibility, and IL-27 gene rs153109 A>G polymorphism carriers have a higher risk of disease in heterozygotes, which is significantly associated with breast cancer. Lu et al. [29] also showed that breast cancer patients had a significantly higher serum IL-27 level than healthy controls, and the serum IL-27 level was closely related to the clinical stage of breast cancer. Furthermore, hormone receptor-positive breast cancer patients have higher levels of IL-27 than hormone receptor-negative patients. Therefore, IL-27 may be a new breast cancer biomarker, providing a new direction for the antitumor effect of IL-27.

In conclusion, our immunohistochemical analysis identified many NK cells in TNBC tissues. Furthermore, we identified associations between CD4+ and CD8+ T cells and the tumor tissue grade, clinical stage, and tumor size, and NK cells and IL-27 expression were related to tumor volume and lymph node metastasis. Our prognostic analysis also demonstrated that NK cell positivity did not affect the OS for three subtypes. However, patients with TNBC who were NK cell-positive had a worse prognosis than those who were NK cell-negative. Overall, the immune system is complex, and many factors affect a patient’s immune status. Therefore, continued exploration of the anti-tumor effects of T cells and NK cells is necessary to improve advanced breast cancer treatments.

Acknowledgments: We thank all patients who participated in this study and donated samples.

Funding

This work was supported by the Natural Science Foundation of Xinjiang Uygur Autonomous Region (Grant No. 2017D01C148).

Competing Interests

All other authors report no conflicts of interest.

Author Contributions

MM conducted the experiments and wrote the manuscript. TZ and ZZ provided research design. TZ And Mayinuer A gave the experimental guidance. PT contributed to the samples collection. HZ designed and directed the whole research. All authors approved the final manuscript.

Ethics Approval

All procedures were performed in accordance with the World Medical Association Code of Ethics (Declaration of Helsinki) for experiments involving humans and approved by the Ethics Committee of the Xinjiang Uygur Autonomous Region People's Hospital (code KY2018011882). This article does not contain any studies performed on animals by any of the authors. Informed consent was obtained from all individual participants included in the study.

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Table 1. Relationship between CD4+ T cells, CD8+ T cells, NK cells and IL-27 and clinical characteristics in patients with luminal A type breast cancer

Luminal A		N	positive	negative	x²	P	positive	negative	x²	P	positive	negative	x²	P	positive	negative	x²	P
			CD4				CD8				NK				IL27
Age (year)	≤52	27	20	7	0.527	0.468	19	8	0.077	0.781	15	12	0.483	0.487	22	5	0.415	0.520
Age (year)	＞52	23	19	4			17	6			15	8			17	6
Histologic grade	I	7	3	4	6.032	0.049	4	3	0.929	0.629	3	4	0.996	0.608	4	3	-	0.212
	II	40	33	7			30	10			35	15			32	8
	III	3	3	0			2	1			2	1			3	0
Clinical stage	I	23	16	7	1.894	0.388	13	10	7.669	0.022	14	9	0.941	0.625	17	6	0.117	0.943
	II	22	19	3			20	2			14	8			16	6
	III	5	4	1			3	2			2	3			4	1
Tumor size (cm)	≤2	38	31	7	0.473	0.492	29	9	1.463	0.278	22	16	0.041	0.839	30	8	0	1
Tumor size (cm)	＞2	12	8	4			7	5			8	4			9	3
metastatic lymph nodes	No	36	29	7	0.102	0.749	25	11	0.087	0.768	22	14	0.066	1	28	8	0.004	1
metastatic lymph nodes	Yes	14	10	4			11	3			8	6			11	3

Table 2. Relationship between CD4+ T cells, CD8+ T cells, NK cells and IL-27 and clinical characteristics in patients with luminal B type breast cancer

Luminal B		N	positive	negative	x²	P	positive	negative	x²	P	positive	negative	x²	P	positive	negative	x²	P
			CD4				CD8				NK				IL27
Age (year)	≤52	25	15	10	0.347	0.769	17	8	0.095	1	11	14	4.023	0.085	17	8	0.936	0.520
Age (year)	＞52	25	17	8			18	7			18	7			20	5
Histologic grade	I	3	3	0	1.728	0.473	1	2	6.237	0.041	1	2	3.073	0.219	1	2	4.572	0.080
	II	21	12	9			12	9			10	11			14	7
	III	26	17	9			22	4			18	8			22	4
Clinical stage	I	5	2	3	2.191	0.345	1	4	9.644	0.006	2	3	2.191	0.345	2	3	5.642	0.059
	II	17	8	7			10	7			7	10			11	6
	III	28	20	8			24	4			20	8			24	4
Tumor size (cm)	≤2	23	17	6	1.817	0.241	17	6	0.311	0.758	13	10	0.038	1	19	4	0.917	0.338
Tumor size (cm)	＞2	27	15	12			18	9			16	11			18	9
metastatic lymph nodes	No	21	11	10	2.122	0.232	12	9	2.850	0.091	10	11	1.602	0.253	13	8	2.753	0.116
metastatic lymph nodes	Yes	29	21	8			23	6			19	10			24	5

Table 3. Relationship between CD4+ T cells, CD8+ T cells, NK cells and IL-27 and clinical characteristics in patients with HER-2 type breast cancer

HER-2		N	positive	negative	x²	P	positive	negative	x²	P	positive	negative	x²	P	positive	negative	x²	P
			CD4				CD8				NK				IL27
Age (year)	≤52	23	13	10	1.898	0.255	19	4	0	1	13	10	1.705	0.239	18	5	0.071	0.790
Age (year)	＞52	27	10	17			22	5			20	7			23	4
Histologic grade	I	6	4	2	0.930	0.654	3	3	2.973	0.226	3	3	0.968	0.703	4	2	4.508	0.105
	II	17	11	6			13	4			12	5			12	5
	III	27	14	13			25	2			18	9			25	2
Clinical stage	I	17	9	8	0.378	0.936	14	3	2.857	0.255	10	7	2.464	0.317	13	4	2.263	0.382
	II	17	10	7			12	5			10	7			13	4
	III	16	10	6			15	1			13	3			15	1
Tumor size (cm)	≤2	21	10	11	1.602	0.253	15	6	1.646	0.200	12	9	1.266	0.366	14	7	4.115	0.042
Tumor size (cm)	＞2	29	19	10			26	3			21	8			27	2
metastatic lymph nodes	No	20	10	10	0.876	0.393	13	7	4.748	0.029	10	10	3.803	0.071	14	6	2.038	0.153
metastatic lymph nodes	Yes	30	19	11			28	2			23	7			27	3

Table 4. Relationship between CD4+ T cells, CD8+ T cells, NK cells and IL-27 and clinical characteristics in patients with TNBC type breast cancer

TNBC分组		N	positive	negative	x²	P	positive	negative	x²	P	positive	negative	x²	P	positive	negative	x²	P
			CD4				CD8				NK				IL27
Age (year)	≤52	21	13	8	0.402	0.752	14	7	0.091	1	14	7	2.952	0.086	16	5	1.034	0.309
Age (year)	＞52	24	17	7			17	7			22	2			22	2
Histologic grade	I	8	3	5	5.381	0.100	4	4	3.502	0.199	5	3	1.897	0.467	5	3	3.831	0.088
	II	12	7	5			7	5			10	2			10	2
	III	25	20	5			20	5			21	4			23	2
Clinical stage	I	16	12	4	0.864	0.719	13	3	2.247	0.328	12	4	0.694	0.766	13	3	0.659	0.771
	II	18	11	7			12	6			15	3			16	2
	III	11	7	4			6	5			9	2			9	2
Tumor size (cm)	≤2	26	14	12	3.291	0.070	14	12	4.946	0.026	18	2	3.012	0.083	19	7	-	0.016
Tumor size (cm)	＞2	19	16	3			17	2			18	1			19	0
metastatic lymph nodes	No	29	17	12	1.467	0.226	16	11	3.528	0.060	20	9	-	0.017	22	7	2.921	0.087
metastatic lymph nodes	Yes	16	13	3			13	3			16	0			16	0

Natural killer cells are associated with prognosis in triple-negative breast cancer patients

Status:

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Abstract

Figures

Introduction

Methods

Results

Discussion

Declarations

References

Tables

Status:

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