Prognostic Significance of Fibrotic Focus and Tumor Infiltrating Lymphocytes in Breast Cancer According to Molecular Subtypes

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Abstract

INTRODUCTION

Breast cancer is a heterogeneous disease with distinct molecular features and different treatment responses. The tumor microenvironment (TME) comprises a network of tumor cells, blood vessels, lymphatics, myofibroblasts, fibroblasts, neuroendocrine cells, adipose cells, immune-inflammatory cells including myeloid-derived suppressor cells, tumor-associated macrophages, neutrophils, tumor-infiltrating lymphocytes (TILs), and T-cells, as well as the extracellular matrix (ECM) [1-4]. The tumor and TME interact closely. This interaction stimulates the heterogeneity of cancer cells and influences tumor progression and metastasis [3,4]. Further, the TME affects the therapeutic response and resistance of the tumor [3,4]. In recent years, the TME has been investigated as a therapeutic target in cancer treatment [4,5].

Cancer-associated fibrosis is an important component of the TME [6]. Various mechanisms including cellular interactions, immune modulation, and ECM remodeling, have been reported to be associated with fibrosis and impact tumor initiation and progression [6]. Fibroblasts within the TME are referred to as cancer-associated fibroblasts (CAFs), and these contribute to cancer-related fibrosis [6-9]. CAFs are known to influence cancer progression by secreting pro-inflammatory proteins, growth factors, and angiogenic factors, thus altering the ECM, and affecting the immune system [6,7]. Further, fibrosis within the TME promotes tumor growth by altering blood flow, enhancing tumor cell intravasation, and enhancing metastasis [6,10,11].

In addition to CAFs, immune cells play an important role in cancer. Within the TME, immune cells regulate the ECM, secrete several pro-tumorigenic and pro-metastatic factors, and control the immune system [7]. Among immune cells, TILs are lymphocytes that invade the tumor tissue and are implicated in recognizing and killing cancer cells [12]. TILs have been described in several solid tumors, including breast cancer [12,13]. Recent studies have shown the prognostic and predictive importance of TILs in breast cancer [13-17].

Although fibrosis and the immune system influence each other within the TME, few studies have examined the relationship between fibrosis and TILs in breast cancer. In this study, we analyzed the association between fibrotic focus (FF), characterized by a mixture of fibroblasts and various amounts of collagen fibers [18] and TILs in breast cancer tissues. We also aimed to determine the prognostic significance of FF and TILs in breast cancer according to its molecular subtypes.

METHODS

This study included patients who underwent surgical treatment for breast cancer at Daegu Catholic University Hospital between 2007 and 2010, and for whom tissue samples were available. The medical records of patients were reviewed retrospectively, and clinicopathological features were evaluated. Molecular subtypes of breast cancer were classified based on the immunohistochemical findings for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 labeling index. Tumor stage was assessed according to the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual for breast cancer. The informed written consent was obtained from all patients for the use of their data. The ethics review of the study was waived by the Institutional Review Board at Daegu Catholic University Hospital according to the deliberation criteria (CR-18-009).

Formalin-fixed and paraffin-embedded (FFPE) specimens from the patients were stained with hematoxylin and eosin, and the pathological findings were reviewed by an experienced pathologist. Immunohistochemistry (IHC) assays were performed on FFPE specimen sections using the Bond Polymer Intense Detection system (Leica Microsystems, Mount Waverley, Australia) according to the manufacturer’s instructions. ER and PR statuses were considered positive at an Allred score of ≥ 3. HER2 expression was classified as 0, 1+, 2+, and 3+ by IHC according to the updated 2018 American Society of Clinical Oncology/College of American Pathology (ASCO/CAP) guidelines for HER2 [19], and in situ hybridization was performed for IHC 2+ equivalent. The Ki-67 labeling index was expressed as a percentage and was graded as ‘positive’ if the number of positive cells was ≥ 20%. Bcl-2, p53, and epidermal growth factor receptor (EGFR) were further categorized into four groups: 0 (< 5%), 1+ (5%-9%), 2+ (10%-49%), and 3+ (≥ 50%) using IHC assay; 1, 2, and 3 were designated as positive and 0 as negative according to the College of American Pathologists guidelines. FF were characterized as a scar-like area or radially expanding fibrous bands consisting of fibroblasts and collagen fibers within the tumor, and were surrounded by a highly cellular zone of infiltrating carcinoma cells (Figure 1) [20]. FF-positivity was defined as FF of 1 mm or more showing characteristics of FF, with fibroblasts arranged in irregular or storiform patterns showing increased fibroblast cellularity and/or collagenization [20]. The percentage of stromal TILs was assessed semi-quantitatively as 0 (no or scant lymphocytes), 1 (0%-10%), 2 (11%-49%), and 3 (50%-100%); 1, 2, and 3 were designated as positive and 0 was designated as negative (Figure 2). A tissue microarray (TMA) was constructed for the breast tissues following the methods described in our previous study [21]. Additional IHC assays on TMA sections were performed using antibodies against cluster of differentiation (CD)4 (Leica Biosystems, Wetzlar, Germany), CD8 (Dako, Glostrup, Denmark), and CD68 (Dako). The total number of CD4+ T cells, CD8+ T cells, and CD68+ macrophages was counted in the stroma and cancer cell nests for each tissue core under a microscope.

Figure 1.

Representative histology of fibrotic focus (FF) in breast cancer. Arrows indicate the area of FF in invasive breast cancer tissues (H&E; magnification, × 100).

Figure 2.

Representative histology of tumor-infiltrating lymphocytes in breast cancer. (A) Negative expression, (B) Positive expression (immunohistochemical staining; magnification, × 100).

Statistical analysis was performed using SPSS Statistical software (version 25.0; IBM, Armonk, USA). The association of FF and TILs with clinicopathological variables was analyzed. The chi-square test or Fisher’s exact test was used to compare categorical variables, and Student’s t-test or the nonparametric Mann-Whitney U-test was used for continuous data. Multivariate analysis was performed for variables found to be significant in the univariate analysis. Survival data were analyzed using Kaplan-Meier survival analysis. All tests were two-sided. A p-value < 0.05, was considered to indicate statistical significance.

RESULTS

Of the 71 patients included in this study, 69 had invasive ductal carcinoma not otherwise specified (97.2%), one had papillary carcinoma (1.4%), and one had mucinous carcinoma (1.4%). The mean age of the patients was 53.4±12.9 years (range, 34-90 years). One male patient with breast cancer was included in this study. Most patients had early-stage breast cancer (stage I and II), accounting for 90.1%, and the remaining patients had stage III (7.0%) and stage IV (2.8%). Among the molecular subtypes of breast cancer, the luminal B subtype accounted for the most (42.3%), followed by the luminal A subtype (25.4%), HER2 subtype (21.1%), and basal type (11.3%). The clinicopathological characteristics are presented in Table 1.

Table 1.

The clinicopathological characteristics of patients (n=71)

FF were present in 31.3% of patients, and TILs were present in 81.7% of the patients. FF and TIL levels showed a positive correlation (p= 0.049). FF was significantly associated with tumor size (p< 0.001), lymphovascular invasion (p< 0.001), regional lymph node metastasis (p< 0.001), and tumor stage (p= 0.002) (Table 2). Multivariate analysis showed that large tumor size and positive lymph node metastasis were predictive factors for the presence of FF (p = 0.007 and p = 0.008, respectively) (Table 3). We determined the area under the curve (AUC) of the receiver operating characteristic curve of the mean tumor size for FF. For the cut-off value of 1.95 cm, the AUC was 0.785, with a sensitivity of 81.0% and a specificity of 65.2% (p< 0.001).

Table 2.

Association of fibrotic focus and TILs with clinicopathological characteristics of breast cancer

Table 3.

Univariate and multivariate analysis of association between fibrotic focus and clinicopathological characteristics

TILs were significantly associated with menopausal status (p= 0.039), histologic grade (p= 0.009), tubule formation (p= 0.019), nuclear grade (p= 0.035), mitosis (p= 0.007), HER2 overexpression (p= 0.023), and molecular subtype of breast cancer (p= 0.001) (Table 2). Further, TILs were associated with the number of CD8+ T cells (p= 0.001) (Table 2). Multivariate analysis showed that postmenopausal status, luminal B subtype, and HER2 subtype were associated with the presence of TILs (p= 0.011, p= 0.005, and p= 0.006, respectively) (Table 4). When stratified by TILs, FF were significantly associated with tumor size, regional lymph node metastasis, extranodal extension, lymphovascular invasion, tumor stage, recurrence-free survival (RFS), and overall survival (OS) in TIL-positive cases (p< 0.001, p= 0.005, p= 0.005, p< 0.001, p= 0.014, p= 0.020, and p= 0.008, respectively), whereas no significant association was found in TIL-negative cases (Table 5).

Table 4.

Univariate and multivariate analysis of association between TILs and clinicopathological characteristics

Table 5.

Association between fibrotic focus and clinicopathological characteristics of breast cancer according to the status of TILs

We then compared the clinicopathological data according to HER2 overexpression status and analyzed the association between TILs and clinicopathological features of breast cancer based on HER2 status. In HER2-negative breast cancer, TILs were significantly associated with tumor size, tumor necrosis, histologic grade, ER status, and EGFR expression (p= 0.038, p= 0.014, p= 0.012, p= 0.014, and p= 0.006, respectively), whereas no significant correlation was found in HER2- positive breast cancer.

The median follow-up period was 98.3 months (range 1-158 months). Among the total patients, there were 10 recurrences (14.1%), nine breast cancer-related deaths (12.7%), and three non-cancer-related deaths. The 10-year RFS rate was 82.9% and 10-year OS rate was 86.3%, respectively. The OS in patients with FF was significantly shorter than that in patients without FF (p = 0.021) (Figure 3B). The RFS of FF-positive patients tended to be lower than that of negative patients, but the difference was not statistically significant (p= 0.055). When compared according to the status of HER2 overexpression, the OS and RFS were significantly associated with FF in HER2-negative breast cancer (p = 0.030 and p = 0.018, respectively) but not in HER2-positive breast cancers (Figure 4). No significant association was found between TIL levels and patient survival.

Figure 3.

Association of fibrotic focus (FF) and tumor infiltrating lymphocytes (TILs) with patient outcome. (A) Recurrence free survival according to FF, (B) Overall survival according to FF, (C) Recurrence free survival according to TILs, (D) Overall survival according to TILs.

Figure 4.

Association between fibrotic focus (FF) and patient outcomes according to HER2 status. (A) Recurrence free survival according to FF in HER2-negative breast cancer, (B) Overall survival according to FF in HER2-negative breast cancer, (C) Recurrence free survival according to FF in HER2-positive breast cancer, (D) Overall survival according to FF in HER2-positive breast cancer. HER2 = human epidermal growth factor receptor 2.

DISCUSSION

Fibrotic and immune microenvironments are important for controlling breast cancer development and progression [7]. In recent years, the prognostic significance of FF and TILs in breast cancer has been described [13-18,22-24]. Yanai et al. [23] reported that the presence of FF was significantly associated with low/intermediate TILs in the stroma of triple-negative breast cancer. Shimada et al. [24] showed an association between FF and tumor-associated macrophage infiltration in breast cancer. However, there are few studies on the relationship between FF and TILs in breast cancer. In this study, we showed that FF are positively associated with TILs in breast cancer. Further, in TIL-positive cases, FF were significantly associated with poor prognostic factors including larger tumor size, regional lymph node metastasis, lymphovascular invasion, and poor RFS and OS. Our results thus suggest an interaction mechanism between TILs and FF that influences breast cancer progression. Carcinogenesis is similar to the chronic inflammatory process in mechanisms such as dysregulated cell proliferation and altered immune infiltration [6]. Chronic inflammation results in fibrosis, which predisposes to cancer initiation and contributes to the development of CAFs [6]. In this regard, TILs are implicated in the formation of fibrosis in the TME and affect cancer progression. However, further studies are required to elucidate the mechanism by which TILs and FF interact in breast cancer.

The prognostic significance of TILs in breast cancer according to subtype has been well recognized. TILs have been found to be more abundant in ER-negative/HER2-negative and HER2-positive breast cancers than in ER-positive/HER2-negative tumors [25]. Further, TILs have been shown to be associated with a better prognosis in ER-negative/HER2-negative and HER2-positive breast cancer [25-28]. In our study, multivariate analysis showed that TILs were associated with the HER2 subtype. We also analyzed the association between TILs and the clinicopathological features of breast cancer based on HER2 status. In HER2-negative breast cancer, TILs were significantly associated with larger tumor size and higher histologic grade, whereas no significant correlation was found in HER2-positive breast cancer. No prognostic significance of TILs was found according to the molecular subtypes of breast cancer in this study. These inconsistent results may be due to the small sample size used for analysis, as this study included only eight triple-negative breast cancers and 15 HER2-positive breast cancers.

Unlike TILs, the results of studies investigating the association between FF and the molecular subtypes of breast cancer are inconsistent. Hasebe et al. [29] and Li et al. [30] showed a positive correlation between FF and HER-2 protein overexpression in breast cancer. Mujtaba et al. [22] reported that FF were negatively associated with HER-2 expression and positively associated with hormone receptor (HR) expression. In our study, FF was not related to HR expression or HER2 overexpression.

In breast cancer, FF are associated with poor prognostic factors [18,20,22,29,30]. Previous studies have demonstrated that the presence of FF is associated with larger tumor size, higher histologic grade, higher tumor cell proliferative activity, necrosis, higher stage, lymph node metastasis, and vascular and lymphatic vessel invasion [18,20,29,30]. Survival analysis showed shorter disease-free survival, RFS, and OS in patients with breast cancer positive for FF than in those without FF [18,22,29]. Consistent with previous studies, our results showed that FF were associated with poor prognostic factors including larger tumor size, lymphovascular invasion, and positive lymph node metastasis. In addition, the OS of FF-positive patients was significantly shorter than that of FF-negative patients in this study.

Our study has several limitations. First, this was a retrospective study, and selection bias may have influenced the results owing to the limitations of available data. Further, patient prognosis may vary depending on the choice of treatment methods. Second, our study included a relatively small number of patients with breast cancer. A small sample size may weaken the statistical power and limit the generalizability of the study results. Therefore, further well-planned prospective studies with a larger cohort are required to validate our findings. Despite these limitations, our study is valuable as it provides additional meaningful information regarding the association between FF and TIL in breast cancer, which is relatively unclear.

In conclusion, our study showed an association between FF and TILs in breast cancer. FF were positively associated with TILs in breast cancer. In TIL-positive cases, FF were significantly associated with poor prognostic factors for breast cancer and poor OS. However, further studies are needed to elucidate the interactions between FF and TILs in breast cancer.

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