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Abstract
Background: Galectin-3 has an important role in metastasis, therefore, Galectin-3-focused therapies have attracted attention for various cancers. Aim: We aimed to reveal the relationship between the expression of Galectin-3 within the tumor/cancer-associated fibroblasts (CAF) and clinicopathological parameters in patients with invasive ductal carcinomas. Materials and Methods: Hematoxylin and eosin-stained slides of breast excision materials diagnosed between 2010 and 2016 were re-examined retrospectively. Accordingly, 118 cases (luminal group = 58, Human epidermal growth factor receptor 2 (HER2) group = 27, and triple-negative breast carcinoma group [TNBC] =33 cases) were included. Galectin-3 levels were evaluated with a calculated H-score in tumor and semiquantitatively in CAFs. Statistical Analysis: Data was analyzed with t-tests and Chi-square tests. Kaplan–Meier and Log-rank tests were used for survival analysis. Results: The presence of Galectin-3 expression in CAFs but not in the tumor was associated with the greater number of axillary metastatic nodes and advanced pN stage. The loss of Galectin-3 expression in CAFs was more frequent in TNBC. There was no significant relationship between the expression level of Galectin-3 and survival status. However, in most of the cases with distant metastasis or patients who died, Galectin-3 was negative in the tumor, whereas it was positive in CAFs. Conclusions: The expression of Galectin-3 in tumors and CAFs may have a role in metastasis to axillary lymph nodes and distant sites. In terms of molecular subtype, TNBCs show a relationship with Galectin-3 negativity in CAFs.
Keywords: Cancer-associated fibroblast, Galectin-3, invasive ductal carcinoma, metastasis, molecular subtypes
How to cite this article: Introduction 
Breast carcinoma includes a heterogeneous group of diseases in terms of histopathological appearance, molecular features, and biological behavior. Molecular subtyping is based on immunohistochemical staining of the estrogen receptor (ER), progesterone receptor (PR), HER-2 expression status, and the Ki-67 proliferation index in routine practice.[1] Different subtypes differ in terms of metastasis potential/mechanisms, treatment options, and prognosis.[2],[3] We now know that in addition to the tumor cells themselves, the tumor microenvironment in which they are located has an important effect on shaping these differences.[4]
The tumor microenvironment includes cancer-associated fibroblasts (CAFs), immune system cells, endothelial cells, and noncellular elements found in the extracellular matrix. CAFs constitute the predominant component.[5] CAFs shape the tumor microenvironment through cytokines and growth factors that they secrete and with the help of epigenetic changes. In addition, in studies investigating gene expression profiles in CAFs and normal fibroblasts, different expression levels were found in genes encoding enzymes such as matrix metalloproteinases and protease inhibitors. This exhibits that CAFs are programmed to increase the migration and invasion capacity of tumor cells.[6],[7],[8]
Different molecular subtypes have been found to contain different CAF groups based on these functions of CAFs in breast carcinoma studies. The different CAF groups are also reported to determine the metastasis region/time and the prognosis, which varies among molecular subtypes.[4],[9] Once CAFs were understood to play an important role in all stages of tumorigenesis, the development of treatment options targeting this particular component was emphasized. It has also been shown that CAFs are responsible for tumor resistance to chemotherapeutic drugs.[10],[11],[12]
Galectin-3 belongs to a family of carbohydrate-binding proteins with a high affinity for β-galactosides.[13] It is expressed by epithelial cells, activated macrophages, and some neurons in normal tissues.[13] It can be transported between the nucleus, cytoplasm, and mitochondria within the cell and can also be present in the extracellular matrix.[13] It is involved in many physiological and pathological processes, both inside and outside the cell, thanks to this wide distribution.[14],[15] These include the regulation of apoptosis and signal transduction, gene expression and mRNA regulation, immune regulation, cell adhesion, cell motility, and angiogenesis.[13],[14],[15] All these reported functions are important features at every stage of the tumorigenesis process, from invasion to metastasis. However, the last three (cell adhesion, cell motility, and angiogenesis) are particularly important as they play a major role in the cancer metastasis mechanism.[13]
Galectin-3 has also been reported to be expressed from cancer-related fibroblasts in recent years.[14],[15],[16] Therefore, Galectin-3-focused therapies have attracted attention in targeted therapy studies for various solid cancer types, and it could potentially be a useful marker, especially in the prevention of resistance to some chemotherapeutic drugs.[17],[18],[19]
We aimed to study the Galectin-3 immunostaining features in tumor cells and the stromal fibroblasts surrounding them, and thus reveal its relationship with molecular subtypes and clinicopathological parameters, and also to investigate the relationship between Galectin-3 expression status in tumor tissue/CAFs and survival (disease-free survival, overall survival) in patients with invasive breast carcinoma in this study.
Materials and Methods Cases of invasive breast carcinoma diagnosed since 2010 were screened from the intranet system of the hospital. All slides belonging to cases with a diagnosis of invasive ductal carcinoma that did not exhibit a specific histological type were removed from the archive and re-examined retrospectively. Clinicopathological findings (such as age, neoadjuvant treatment history, treatment and follow-up information, nuclear grade, histological grade, tumor size, number of positive lymph nodes, presence of in situ carcinoma, microcalcification and perinodal spread, hormone receptor expression, HER2 expression status, Ki-67 proliferation index) of the patients were recorded. Patients with distant metastasis at the time of diagnosis, patients who received neoadjuvant treatment, cases including only in situ carcinoma, and cases whose clinical follow-up information or blocks could not be accessed were excluded from the study.
Accordingly, a total of 118 cases were included in the study. The molecular subtype distribution of these cases was luminal in 58, HER2 in 27, and triple-negative breast carcinoma (TNBC) in 33 cases.
The grading was performed according to Nottingham combined grade system.[20] pT and pN stages were determined based on the American Joint Committee on Cancer (AJCC) Cancer staging manual 2017.[21]
Immunohistochemical staining was performed with the streptavidin-avidin-biotin method. For each case, the block that reflected the tumor best contained the least necrosis and contained normal breast parenchyma as an internal control was determined to undergo immunohistochemical staining from 10% neutral buffered formalin-fixed paraffin-embedded blocks.
The presence of cytoplasmic and/or membranous staining in normal breast luminal epithelial cells in areas adjacent to the tumor was accepted as a positive control for Galectin-3 (Cell Marque, 9C4, 1/75). For invasive tumor cells, the staining intensity was graded as absent-mild-moderate-severe (0-1-2-3) [Figure 1], [Figure 2], [Figure 3]. The staining intensity was evaluated at 400× magnification in the most intensive area. Staining distribution was evaluated by providing a percentage of all tumoral areas. As a result of multiplying these two values with each other, a value between 0 and 300 (H-score) was found so that “0” represented no staining and “300” represented diffuse staining, both with strong intensity. All cases except those with an H-score of 0 were accepted as positive in terms of Galectin-3 immunohistochemical staining.
Figure 1: Invasive tumor cells show weak immunostaining (score 1) for Galectin-3 at the center of the figure. CAFs, however, display strong staining for Galectin-3
Figure 2: Invasive tumor cells show moderate degree of immunostaining for Galectin-3 (score 2). Galectin-3 staining is negative in the peritumoral CAFs
Figure 3: Invasive tumor cells show severe degree of immunostaining for Galectin-3 (score 3)Fibroblasts located in the intratumoral compartment were appreciated as CAFs. CAFs staining distribution was evaluated as 0 (0%), 1 (<10%), 2 (10–50%), or 3 (>50%) (0-1-2-3) [Figure 1] and [Figure 2]. The groups except 0 were accepted as positive. These percentages were calculated by proportioning the stained CAFs to all CAFs.
Estrogen receptor (Ventana, SP-1, ready-to-use), progesterone receptor (Ventana, 1E2, ready-to-use), and CerbB2 (Ventana, 4B5, ready-to-use) expression status were evaluated immunohistochemically according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) breast cancer guidelines.[22],[23] The Ki-67 (Ventana, 30-9, ready-to-use) staining rate was provided by calculating the percentage of positive staining of tumor nucleus in the area with the most intensive staining at the periphery of the invasive tumor. Besides, the Ki-67 value threshold was accepted as 20% to differentiate low (≤20%) and highly (>20%) proliferating invasive breast carcinomas. Ki-67 staining was performed for 106 cases.
Mean and standard deviation, and median minimum and maximum range values were used as descriptive statistics to define continuous variables, and frequency distribution rates and percentages were used to describe categorical variables. Kolmogorov–Smirnov normality tests were employed to determine the normal distribution of continuous variables. Paired comparisons between groups were investigated using independent samples t-tests and Chi-square tests. Associations between variables were determined via Pearson moment correlation coefficients. Survival analysis was carried out using Kaplan–Meier and Log-rank tests. A value of P < 0.05 was accepted to reflect statistical significance. IBM SPSS 20 was used for the data analysis.
Results A total of 118 invasive ductal carcinoma cases were studied (luminal A group: 6 cases, luminal B group: 52 cases, HER2 group: 27 cases, and triple-negative group: 33 cases). The clinicopathological characteristics of all cases are summarized in [Table 1].
Table 1: Clinicopathological features of the cases with invasive ductal carcinomaEvaluation of the Galectin-3 staining features in the tumor cells of the 118 cases revealed a positive H-score in 48 (40.7%) (H-score: 1–70), and an H-score of 0 (negative) in 70 (59.3%). Although cytoplasmic staining (positive) with Galectin-3 in CAFs was found in 74 cases (62.8%), no staining (negative) was found in 44 cases (37.2%). The staining distribution in cases with positive CAF Galectin-3 staining was under 10% in 45 cases, 10%–50% in 23 cases, and over 50% in six cases.
Evaluation of the relationship between Galectin-3 staining status in the tumor cells and the clinicopathological parameters revealed that the number of metastatic lymph nodes was significantly higher and the pN stage was more advanced in patients without staining. Besides, multicentricity was more common and the mean Ki-67 proliferation index level was significantly lower in patients without Galectin-3 staining in the tumor cells (P < 0.05).
No significant relationship was found between Galectin-3 expression status in tumor cells and the age, nuclear grade, histological grade, tumor size, T stage, presence of angiolymphatic invasion and perineural invasion, multifocality, in situ component presence, microcalcification, perinodal spread, molecular subtype distribution, metastasis, and survival status [Table 2].
Table 2: Distribution of clinicopathological parameters in groups of the tumor with and without Galectin-3 expressionWhen we examined the relationship between the Galectin-3 staining status in CAFs and the clinicopathological parameters, the number of metastatic lymph nodes was significantly higher (P < 0.01), and the pN stage was more advanced (P < 0.001) in patients with Galectin-3 expression as compared with those patients without staining for Galectin-3. In addition, a significant difference was present between the CAF Galectin-3 expression-positive and expression-negative cases in terms of molecular subtype distribution. Accordingly, cases with no Galectin-3 staining in the CAFs were found to be more commonly associated with the triple-negative molecular subtype (P < 0.01) [Table 3].
Table 3: Distribution of clinicopathological parameters in CAF Galectin-3 negative and positive groups of tumorNo significant relationship was found between Galectin-3 expression status in the CAFs and the age, tumor size, nuclear grade, histological grade, T stage, angiolymphatic invasion, perineural invasion, multifocality multicentricity, in situ component, microcalcification, perinodal spread, Ki-67 proliferation index, metastasis, and survival status (P > 0.05).
The median follow-up duration was 71 months (3–113 months) and 28 cases died during follow-up. In the 28 cases that died, immunostaining for Galectin-3 in tumor cells was present in 11 cases and absent in 17 cases, whereas immunostaining in the CAFs was present in 18 cases and absent in 10 cases. Distant metastasis was detected in 23 patients and axillary metastasis in one patient. Metastasis areas were the bone (10), lung (7), brain (6), liver (3), other breasts (1), axilla (1), and mediastinum (1). The median time to metastasis was 34 months (12–67 months). Galectin-3 staining features in the cases who died and/or experienced distant metastases are summarized in [Table 4].
Table 4: Analysis of the cases in terms of distant metastasis and survival according to Galectin-3 expression statusNo significant relationship was found between the Galectin-3 expression status in tumor cells and CAFs and the disease-free survival (DFS) or overall survival (OS) (P > 0.05) when evaluated with the Kaplan–Meier method.
Discussion We aimed to investigate Galectin-3 expression in tumor cells and CAFs in breast carcinoma cases diagnosed with invasive ductal carcinoma without a specific histological type and to study its relationship with other clinicopathological parameters, mainly the molecular subtype and survival status. Accordingly, we found that expression loss of Galectin-3 in invasive tumor cells and the presence of Galectin-3 expression in CAFs exhibited a higher number of metastatic axillary lymph nodes (and more advanced pN stage). Besides, cases with expression loss of Galectin-3 in tumor cells showed a lower Ki-67 proliferation index compared with those of cases with Galectin-3 expression. In addition, a significant relationship was present between the Galectin-3 expression status in CAFs and the molecular subtype of the tumor. The incidence of the triple-negative molecular subtype was higher in cases with no Galectin-3 expression in CAFs. No statistically significant relationship was found between the Galectin-3 expression status and the disease-free survival or overall survival in tumor cells and CAFs.
In many studies of breast carcinoma, relatively lower Galectin-3 expressions have been reported in invasive tumor cells than in normal breast tissue.[18],[24],[25] Conversely, only a few studies have reported increased levels of Galectin-3 expression in cancerous tissues compared with that of normal breast tissue.[17] In our study, we detected strong cytoplasmic immunostaining for Galectin-3 in terminal ductal lobular unit structures representing normal breast tissue and stromal fibroblasts in the nontumoral region. In the study by Castronovo et al.,[24] when the level of Galectin-3 immunostaining was evaluated on a scale of 0-1-2-3, and all intensities of nuclear-cytoplasmic or membranous staining were considered as positive staining, gradually decreasing immunostaining intensities were reported in epithelial cells in normal breast tissue compared with areas of in situ carcinoma and in areas of situ carcinoma relative to areas of invasive carcinoma. In the same study, loss of Galectin-3 staining in tumor cells was observed more frequently in patients with axillary lymph node invasion than in those who did not. Based on these data, the researchers concluded that a decrease in the level of Galectin-3 expression renders the cells an invasive phenotype. Similarly, in our study, we determined that the loss of Galectin-3 expression in tumor cells correlated with a higher number of metastatic axillary lymph nodes and advanced pN stage.
Only a few studies are investigating the relationship between molecular subtypes and the tumor and CAF Galectin-3 expression levels in breast carcinomas. Grosset et al.[18] investigated Galectin-3 levels in the tumor cells and stroma in 213 breast cancer cases with molecular subtyping. Although no significant difference was found between the molecular subtypes and tumoral Galectin-3 expression status, a significantly higher level of Galectin-3 expression in the tumor stroma was found in HER2 and TNBC groups. Although their study is one of the most comprehensive studies, where the level of Galectin-3 in the tumor stroma was investigated immunohistochemically in molecularly subtyped breast cancers, it is not clear which cell component was used to evaluate Galectin-3 in the stroma. Besides, the tissue microarray method was used and the amount of tumor tissue evaluated was limited to a tissue sample of 1 mm. Galectin-3 expression levels in the tumor and surrounding stroma were investigated in invasive lobular carcinomas in another study where Galectin-3 staining was evaluated with an immunohistochemical method in 3 mm tumor tissues obtained with the tissue microarray method.[26] Accordingly, the Galectin-3 expression level in invasive lobular carcinoma cells with triple-negative characteristics was found to be significantly increased compared with those without triple-negative characteristics. Besides, the stromal Galectin-3 expression level in invasive lobular carcinoma with triple-negative phenotype was found to be significantly lower than those of the stroma of invasive lobular carcinomas with no triple-negative phenotype. The researchers did not make any further comment in terms of the effect of Galectin-3 on tumorigenesis based on their current findings.[26] In another study by Zhang et al.,[17] nuclear and/or cytoplasmic staining of 1% and above was accepted as positive in full-thickness tissue sections containing the tumor, and the level of Galectin-3 expression in tumor cells was found to be significantly higher in TNBCs. Some studies did not find a relationship between Galectin-3 expression in tumor cells and the hormone receptor expression and/or HER2 expression status.[25],[27] We found a significant loss of Galectin-3 expression in CAFs in the TNBCs in this study, in which we evaluated full-thickness tumor tissue sections. However, no significant difference was present among the molecular subtypes regard with Galectin-3 expression levels in tumor cells.
There are a few studies that search the relationship between the Galectin-3 expression status and histopathological parameters in CAFs. A total of 273 breast carcinoma cases were evaluated after division into two groups as absent/mild and moderate/severe in terms of Galectin-3 expression in stromal fibroblasts in the study by Moisa et al.[28] The cases that showed moderate/severe stromal CAF Galectin-3 expression were found to have a more advanced pN stage. However, no significant relationship was seen between the tumor/stromal Galectin-3 expression, and the number of metastatic lymph nodes and pN stage in the study by Grosset et al.[18] A higher Ki-67 proliferation level was also reported in the cases with Galectin-3 expression in CAFs in this latter study. We did not find a relationship between Galectin-3 staining status and Ki-67 level in CAFs in our study. However, a significantly higher Ki-67 proliferation index was present in cases with Galectin-3 expression in tumor cells.
Few studies are investigating the relationship between Galectin-3 expression status in tumor cells and histopathological parameters (number of metastatic lymph nodes, pN stage, Ki-67 proliferation index, etc.) in breast carcinomas and the findings of these studies are also contradictory. Although a significant relationship was found between tumor Galectin-3 expression and the number of metastatic lymph nodes and pN stage,[17] some studies did not find such a relationship.[18],[24],[27],[28] Similarly, we found that cases with Galectin-3 expression in CAFs but not in tumors had a higher number of metastatic lymph nodes and a more advanced pN stage. This data and our finding of more common CAF Galectin-3 negativity in the TNBC group are somewhat complementary. The reason is that while the luminal type of breast carcinomas lead more commonly to axillary lymph node metastasis with lymphovascular invasion, TNBCs tend to cause distant organ metastases (such as lung and brain) in general.[3] This supports the contribution of CAFs to tumor metastasis mechanisms. We found a significant expression loss for Galectin-3 in CAFs in the TNBC group in this study. In addition, although Galectin-3 expression was not observed in the tumor in most of the patients with distant metastasis who died, Galectin-3 expression was present in the CAFs. Based on these findings, we believe that Galectin-3 may have a role in the mechanisms of tumor metastasis.
There are also studies that investigated the relationship of Galectin-3 expression levels in the tumor cells and stroma with the prognosis. Logullo et al.,[29] investigated the Galectin-3 immune expression in 92 early-stage breast carcinoma cases and they accepted the presence of cytoplasmic and/or nuclear staining of 10% or more in the tumor cells and stromal component as a threshold value. They found that there was no association between Galectin-3 immune expression in the tumor cells/stroma and disease-free survival (DS) or overall survival (OS). However, it was not clearly specified which cellular component in the stroma was stained with Galectin-3. We did not find a significant relationship between Galectin-3 expression levels in tumor cells/CAFs and the DFS/OS in our own study. In contrast, some publications revealed that increased Galectin-3 expression levels in tumor cells had an association with good or poor prognosis.[18],[25],[27] The results of the few studies investigating the relationship of Galectin-3 expression in the stroma with the prognosis are similarly conflicting. Although some have reported a worse prognosis in cases with increased stromal Galectin-3 expression, others have not found any relationship.[17],[18],[28]
In the current study, we noted that among the cases with distant metastasis, tumoral Galectin-3 negativity was observed in 71.4% of the cases and CAFs positivity in 60.7%. Among the cases that died, tumoral Galectin-3 negativity was observed in 60.7% of the cases and CAFs positivity in 64.2%. The most common area of metastasis was the bone, and Galectin-3 expression was negative in the tumor in all cases with bone metastasis (n = 10), but positive in the CAFs in 60%.
Conclucion In this study, we investigated 118 breast carcinoma cases, diagnosed as invasive ductal carcinoma, and with a median clinical follow-up of 71 months, in terms of Galectin-3 immune expression in tumor cells and CAFs. The low H-score level, which we used to indicate positivity for Galectin-3 in tumor cells is a limitation of this study. In recent years, the data that Galectin 3 can be a potential agent in targeted therapies as well as its effect on drug resistance are exciting in terms of elucidating the relationship of Galectin-3 with tumorigenesis and metastasis.
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Conflicts of interest
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References 
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CheckDOI: 10.4103/ijpm.ijpm_284_21
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