Soluble IL-2R as a predictor of familial breast cancer

Background: The incidence of breast cancer has been increasing annually, and breast cancer-related diseases, such as breast cancer in the young, ovarian cancer, prostate cancer, and pancreatic cancer, have clearly and steadily increased in number and have become common among the family members of patients with breast cancer. Accordingly, an increase in the incidence of familial breast cancer (FBC) is anticipated in the future. Interleukin (IL)-2 is one of the cytokines that activate CTLs, which are important for cancer immunity. To search for the markers of increased risk for FBC, we examined the sIL-2R levels and immunologic factors in patients with breast cancer and nonfamilial breast cancer (NFBC). Methods: Of the 106 untreated breast cancer patients who gave consent to participate in this study, 24 had FBC and 82 had NFBC. There were 11 healthy individuals included in this study. Serum and peripheral blood mononuclear cells were collected from all patients for the measurement of the levels of sIL-2R, IL-10, VEGF, IL-17, Tregs, and MDSC. Prognosis was assessed and compared, according to the sIL-2R levels (low vs. high). Tissue samples from postoperative patients with high sIL2 were stained with PD-LI and CD8. Results: The sIL-2R level was significantly higher and had significantly better correlations with IL-10, VEGF, IL-17, Tregs, and MDSC levels in FBC than in NFBC. In cases with high sIL-2R level, the Tregs and MDSC levels were significantly higher and the OS and DFS rates were significantly worse in FBC than in NFBC. Among the FBC cases with high sIL-2R level, triple-negative breast cancer tissues stained well for PD-LI and CD8. Conclusions: Compared with NFBC, FBC was associated with higher sIL-2R levels, Th2 significance, and less aggressive cancer immunosuppressive cells. We have identified sIL-2R as a biomarker that can predict the prognosis of FBC. The ability to prospectively on a using FBC: familial breast cancer; NFBC: non-familial breast cancer; HBOC: hereditary breast and ovarian cancer; IL: interleukin; IL–2R: interleukin–2 receptor; sIL–2R: soluble interleukin–2 receptor; VEGF: vascular endothelial growth factor; Treg: regulatory T cell; MDSC: myeloid-derived suppressor cell; CTL: cytotoxic MMR: PD-L1: programmed programmed

3 identify patients who are less likely to have NFBC is a vital step in improving the overall survival of this population.

Background
Familial breast cancer (FBC) is a cluster of breast cancer patients within a family. Most cases of breast cancer occur sporadically in individuals with little to no family history of the condition. Approximately 5% to 10% of breast cancer cases are considered hereditary through an autosomal dominant mechanism (NIH National Center for Advancing Translational Sciences, GARD Genetic and Rare Diseases Information Center, https://rarediseases.info.nih.gov/diseases/10415/familial-breast-cancer). However, the diagnostic analysis of breast cancer-related genes, such as the hereditary breast and ovarian cancer gene, in patients and carriers, as well as the genetic testing of every single family member remain very difficult to perform in a hospital setting. Furthermore, an increase in the prevalence of FBC is anticipated in the future. Regardless, the fact that the number of young breast cancer patients has increased and that patients with recurrent breast cancer require prompt treatment remain.
Interleukin (IL)-2, which is one of the most important cytokines for lymphocyte development, proliferation, and function, is produced by helper cells that differentiated from naïve cells upon stimulation with interferon γ (IFN-γ) or IL-12. The actions of IL-2 include proliferation and activation of T cells, promotion of proliferation and antibody production of B cells, activation of monocytes/macrophages, and proliferation/activation of natural killer cells, to name a few [1]. In addition, IL-2 has been believed to be required for the maintenance of regulatory T cells (Tregs), which release the inhibitory cytokine IL-10 and exhibit immunosuppressive effects [2]. Breast cancer patients have been known to have increased blood levels of IL-2 and its soluble IL-2 receptor (sIL-2R) [3][4][5][6]. In this study, we examined the role of sIL-2R in patients with FBC.

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Cases of FBC probably possess a functional failure of the mismatch repair (MMR) gene, such as the BRCA1/2. A deficient DNA mismatch repair function due to MMR gene mutation increases the number of somatic gene mutations and tumor mutational burden (TMB) and leads to the release of neoantigens from cancer cells that have a large number of gene mutations. Dendritic cells incorporate and degrade neoantigens, which are long peptides that bind to Human leukocyte antigen (HLA) class II and are presented on the cell surface.
Neoantigens are recognized by naïve cluster of differentiation (CD)4+ T cells and produce IL-2. The simultaneously incorporated and degraded short peptides bind to HLA class I, presented on the cell surface, recognized by naïve CD8+ T cells, and express IL-2R. IL-2 binds to IL-2R to induce differentiation and proliferation of cytotoxic T lymphocytes (CTL), which are cancer cell-specific immune cells. Upon activation, CTLs express immune checkpoint molecules, such as programmed cell death 1 (PD-1), on the surface. In addition, due to the IFN-γ produced by CTLs for cancer cell attack, cancer cells express programmed cell death ligand 1 (PD-L1) and suppress CTL activity.
Eventually, CTLs become immune tolerant and incapable of functioning. In such cases, identification of the genes associated with increased TMB would enable the prediction of the sensitivity to immune checkpoint inhibitors and the more efficacious therapy for FBC.

Study subjects
The present study enrolled 11 healthy volunteers and 106 preoperative patients who had FBC was defined as a proband with breast cancer and (1) histologically proven breast cancer or ovarian cancer in at least 3 relatives, 1 of whom should be a 1st degree relative, including the mother or a sister, and with bilateral breast cancer, prostate cancer, and pancreatic cancer in the family or 2) at least 2 successive generations diagnosed as breast or ovarian cancer in at least 1 of the relatives at an age younger than 45 years and with bilateral breast cancer, prostate cancer, and pancreatic cancer in the family. NFBC was defined as only the proband having breast cancer.
After collection of blood samples from the study population, a total of 1 × 10 6 peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll density gradient centrifugation method; aliquots of PBMCs were cryopreserved in a freezing medium.
Plasma was separated by centrifugation and stored at 80 °C until flow cytometry analysis.

Cytokine production
The serum concentrations of sIL-2R, IL-10, VEGF, and IL-17 in the supernatants were measured using as enzyme linked immunosorbent assay kit (Quantikin; R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's protocol.

Statistical analysis
All data were presented as mean ± standard deviation. Differences between groups were determined using Student's t-test. Relationships between 2 variables were quantified using Spearman's rank correlation coefficient test. For the assessment of overall survival (OS) and disease-free survival (DFS), data that were available until the last follow-up date or at 2,500 days were censored. The prognoses of the patients were analyzed using Kaplan-Meier method, and the log rank test was used to determine the univariate significance of the variables. Multivariate Cox regression analysis of the survival of patients with preoperative breast cancer was performed, according to the tumor molecular subtype and Ki67 status, as defined in the NCCN Clinical Practice Guidelines in Oncology [7,8]. Cox proportional hazards model was used to examine the simultaneous effects of multiple covariates on survival. The effect of each variable was described by the hazard ratio, with 95% confidence interval. A p value of <0.05 was considered to indicate statistical significance. SAS software version 9.2 (SAS Institute Inc., Cary, NC, USA) was 7 used for statistical analysis.

Staining for PD-L1 and CD8
Slides were deparaffinized in toluene, rehydrated in graded alcohols, then heat-induced epitope retrieval was performed, followed by a CD8 IHC protocol on clone (C8/144B)Monoclonal Mouse (Dako, Agilent Technologies, Santa Clara, CA) and a PD-L1 IHC protocol on clone E1L3N Rabbit (Cell Signaling Technology, Danvers, MA).

Results
The present study included a total of 106 patients; of these 24 had FBC and 82 had NFBC (Table 1) The sIL-2R level was classified as high or low, based on a cutoff value of 700 U/mL, high 8 serum sIL-2R levels in FBC patients significantly increased the levels of Tregs (p = 0.0008) and MDSC (p = 0.01) (Fig. 3). The upper limit of 700 U/mL was chosen as the cutoff value for classifying sIL-2R as high or low, based on the median sIL-2R value of 667.50 ± 42.54 U/mL in healthy subjects. sIL-2R survival possibility As shown in Fig. 4, FBC patients who had high sIL-2R levels (>700 U/mL), compared with those with low sIL-2R levels, had significantly worse rates of OS (p = 0.0091) and DFS (p = 0.0038).
The Kaplan-Meier plot of DFS was dichotomized, based on sIL-2R expression above and below the median value of 700 U/mL. The Kaplan-Meier method was used to obtain the survival curves, which were analyzed by the log-rank test.

sIL-2 immunostaining
As shown in Fig. 5, immunohistochemistry of the core needle biopsy specimens from the FBC patients before treatment (T4N1M1 stage IV, ER−, PgR−, HER2−)demonstrated relatively more expressions of PD-L1 and CD8 when the sIL-2R level was high.

Discussion
Breast cancer is a heterogeneous disease that comprises multiple molecular subtypes. In this study, we found no significant difference in tumor molecular subtype between FBC and NFBC patients (Table 1). In several malignancies, serum sIL-2R levels are high, compared with those in healthy individuals. Although sIL-2R is not organ-specific, except for malignant lymphoma, measuring its serum level was shown to be valuable for stage evaluation and monitoring during treatment [9]. Breast cancer patients have been known to have increased serum levels of sIL-2R [10], as well as IL-10, VEGF, IL-17, Tregs, and MDSC [11][12][13][14][15]. Others have reported the correlation of sIL-2R in cancer with IL-10, VEGF, 9 and IL-17 and the relationship between Tregs and MDSC [15][16][17][18].
Consistent with the previously reported results, the results of this study showed increased sIL-2R level in all breast cancer patients, compared with that in healthy subjects. Overall, sIL-2R level was significantly higher in FBC than in NFBC. Compared with NFBC, FBC comprises cancer cells that contain more mutations and release neoantigens and has a microenvironment of CD8+ T cells that express IL-2 receptors and release more sIL-2Rs in Notably, proliferation of CTLs does not guarantee the removal of all cancer cells. IL-2 is a systemically administered treatment that can lead to serious side effects, but decreasing its dose may render it ineffective [19,20].
PD-L1 is expressed in 20% of TNBCs, suggesting PD-L1 as a therapeutic target in TNBCs.
Because PTEN loss is one mechanism regulating PD-L1 expression, agents targeting the PI3K pathway may increase the antitumor adaptive immune responses [21].
In this study, immunohistochemical analysis of untreated preoperative cases confirmed tissue expressions of PD-L1 and CD8 in FBC with high sIL-2R and less CD8 staining in NFBC. These results indicated similar expressions of PD-LI in both FBC and NFBC, but in the case of FBC, Th2>Th1 balance or Tregs and MDSC in cancer microenvironment suppressed, on the way, the expression of CD8+ T cells.Although the direct relationship between IL-2 and PD-L1 is unknown, a combination of IL-2 and PD-L1 inhibitors has been the treatment regimen for dominant infections and cancer [22].

Consent for publication
Written informed consent for the publication of this case report and any accompanying images was obtained from all patients. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no financial or nonfinancial competing interests.   Figure 1 Results of sIL-2R evaluation in healthy volunteers and in patients with FBC + NFBC, FBC alone, and NFBC. The sIL-2R levels were higher in the preoperative patients (FBC + NFBC) than in the healthy volunteers. The sIL-2R level of FBC patients was significantly higher, compared with that of healthy volunteers (*p=0.0414) and NFBC patients (**p=0.0155). Data are represented as mean ± SD. P values were determined using the Student's t-test.

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