Abstract
Context:
Tumor microenvironment is emerging as a critical factor for progression of breast cancer. Tumor-associated macrophages (TAMs) play an important role in promoting tumor growth.
Aim:
This study was aimed at correlation of number density (ND) of TAMs with invasive ductal carcinoma (IDC) grading utilizing an image morphometric technique. We also sought to compare the TAMs and ND in the tumoral area and stromal region. We also explored the relationship between the clinical and pathological prognostic parameters.
Subjects and Methods:
The study included 75 cases of IDC that had undergone modified radical mastectomy. The Institutional Ethics Committee approved the study. Samples were classified as Grade 1, 2, and 3. Cases were graded as per the modified Bloom and Richardson criterion. Mean with standard deviation was calculated for each group. We utilized CD68 and CD163 immunostained sections for determining the ND of TAMs. TAMs were evaluated using computerized digital photomicrograph system with image analyzing software. ND was defined as the number of TAMs in total number of TAMs in five high-power fields/total area of five fields. ND was calculated separately in tumor and tumor stroma (TS). Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2/neu (HER2/neu) were scored in accordance with recommendations. Ki-67 was scored as per the recommended guidelines.
Statistical Analysis Used:
Data were tabulated in Microsoft Excel. SPSS version 20.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. To determine the relationship between macrophage density and clinicopathologic parameters, we used the independent t-test. To determine the differences in the parameters, analysis of variance (ANOVA) was utilized.
Results:
Age of the patients ranged from 34 to 58 years (mean: 55.5). One-way ANOVA between various grades of tumor indicating significant differences in terms of CD68 and CD163 densities in tumor and stroma (P < 0.0001). i.e., significant increased density of CD68 and CD163 was observed in Grade 3 tumor as compared to other two groups. A greater histological grade, ER, PR negative status, and a high Ki-67 index were all associated with TAM ND. There was no relation to HER2/neu status. Result of unpaired t-test indicates increased density in stroma as compared to tumor among various grades of IDC.
Conclusions:
We analyzed images with a software using photographs of the stained slides. This helped in quantitative analysis of TAMs on the CD68 and CD163 stained sections. This approach standardizes and reproducibly counts TAMs per unit area. We found significant difference between the number densities of TAMs in grades of invasive breast carcinoma. There were statistically significant differences in numerical densities of TAMs with ER, PR negativity, and Ki-67. There was no correlation with HER2/neu. Densities of CD68 and CD163 densities are more prevalent in TS as compared to intratumoral region.
Keywords: Image analyzing software, immunohistochemistry, macrophages, morphometry, prognosis, semiquantitative scoring, tumor-associated macrophages, tumor microenvironment
INTRODUCTION
The growth of solid epithelial malignancies is influenced not only by the phenotype of tumor but also by the tumor microenvironment, which controls the aggressiveness, size, rate of progression, and spread of tumor cells through secretary as well as immunological interactions.[1] Studies have explored the tumor microenvironment and its immunological role in breast carcinomas.[2-6] Tumor-associated macrophages (TAMs) control the microenvironment, and thus influence the progression and growth of invasive ductal carcinoma (IDC). The method of detection and counting of TAMs is still being standardized.[1]
TAMs are seen in both tumor stroma (TS) as well as in the tumor nests (TN).[7] They polarize and display two types of phenotypes, M1 or M2[8] M1 macrophages stimulate response which induces hypoxia leading to tumor cell death[9] whereas cytokines with anti-inflammatory action are produced by M2 macrophages[1] and are thus responsible for tumor cell survival and growth.[10,11]
The numerical density of TAMs in IDC can be used to determine the function of microenvironment. We can use specific immunohistochemical markers and image morphometry for accurate quantification and pinpointing the location of TAMs.
They can be stained by CD68 which is a pan-macrophage marker and by CD163 which identifies M2 TAMs.[3,12]
This study was aimed at correlation of number density (ND) of TAMs with IDC histological grading utilizing the image morphometric technique. We also sought to compare the two types of TAMs and their presence and ND in TS and the tumor parenchyma. These were also correlated with the clinicopathological prognostic parameters.
SUBJECTS AND METHODS
The study included 75 specimens of patients who underwent modified radical mastectomy for IDC of the breast at a tertiary care hospital. The Institutional Ethics Committee bearing approval number 100039/EC/Study/ CH(EC)/2021 dated 19 Feb 2021. Informed consent was taken. The study comprised cases of pathologically confirmed invasive ductal carcinoma with complete clinicopathological information. Patients who had preoperative treatment in the form of chemotherapy or radiation were barred from participating in the research.
The medical records of the patients were evaluated for patient and tumor characteristics, clinical and pathological information. Staging of the tumors was assessed using 8th edition of the American Joint Committee on Cancer.[13]
We performed immunohistochemistry (IHC) on two representative blocks from cases on 5-μ sections. Each case was stained with CD68, CD163, Estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2/neu (HER2/neu), and Ki67.
Two pathologists performed histopathological evaluations and cases were evaluated using the modified Bloom and Richardson criteria which included three components comprising tubules, nuclear pleomorphism, and mitotic count.[14,15]
Quantitation of tumor-associated macrophages
A software analysis program was used to quantitatively estimate TAMs in the stained histological sections CD68 and CD163.[16] The software was calibrated using standard scale. The sections were first examined at low power (×100 magnification) to identify three areas “hot spots” showing greatest number of TAMs. For each sample, five high-power fields (×400) were captured in these hot areas. TAMs were counted on the recorded images using the image analyzing software. Total area was 0.289 mm2 in five high-power fields.[16]
TAMs ND = Total number of macrophages in five high-power fields divided by the total area of the five fields.
TAMs ND in tumor (NDT) and stromal macrophages infiltrating stroma were counted as stromal macrophages (NDS).
The image analyzer results were revised manually by the pathologists to confirm the number and density of macrophages.
ER, PR, and HER2/neu were scored as per the recommended guidelines.[17,18]
For Ki-67, counting was done in three randomly selected (×40) high-power fields. Thousand cells were counted for scoring from the invasive edge of the tumor. Scoring was done with the help of digital imaging and morphometry.[19]
Statistical analysis
Data were entered in Microsoft Excel and SPSS version 20.0 was used for statistical analysis (IBM Corp., Armonk, NY, USA). The independent t-test was performed for determining the relationship between macrophage density and clinicopathological characteristics. It was also utilized to study the relationship with tumor grade as well as between NDT (Number Density Tumour) and NDS (Number Density Stroma). To analyze the differences in all the factors tested, an analysis of variance (ANOVA) with a post hoc Turkey Honest Significant Difference test was used.
RESULTS
The clinicopathological variables of the cases were as shown in Table 1 with the patients’ mean age being 55.5 years. Fifty-two (69.3%) cases were classified as Stage III, whereas Stage II comprised 20 (26.7%) cases, and 3 (4%) as Stage I.
Table 1.
Clinical and pathological characteristics in patients
| Mean value of studied parameters and mean and SD for Age and Tumour size for three grades of breast carcinoma | |||
|---|---|---|---|
|
| |||
| Parameters (unit) | Grade 1 (N=25) | Grade 2 (N=25) | Grade 3 (N=25) |
| Age (years) | 53.50 (10.23) | 55.78 (11.79) | 57.25 (10.47) |
| Tumor size (cm) | 2.65 (1.28) | 3.61 (3.61) | 3.71 (1.36) |
| Lymph node metastasis (N) | 6 | 12 | 15 |
| ER positivity | 15 | 18 | 3 |
| PR positivity | 12 | 10 | 3 |
| HER2/neu positivity | 10 | 10 | 6 |
| Ki-67 (%) | 3 | 14 | 30 |
ER: Estrogen receptor, PR: Progesterone receptor, HER2/neu: Human epidermal growth factor receptor 2, SD: Standard deviation. 3, N: Number
Figures 1 and 2a-f shows histological sections stained with CD68 and CD163 for all the grades. Figure 3 shows comparable H and E-stained sections of Grade 1, 2, and 3 IDC. Both figures indicate a greater number of TAMs in the stroma than in the parenchyma. Grade 3 tumors had the highest degree of expression of CD68 and CD163. Table 1 displays the mean value with standard deviation of all the examined parameters
Figure 1.
CD68 in various grades. (a and b) Grade 1, (c and d) Grade 2, and (e and f) Grade 3 tumors, (a-d, ×200; e-f, ×400; tumor nests-a, c, e; tumor stroma-b, d, f)
Figure 2.
CD163 in various grades. (a and b) Grade 1, (c and d) Grade 2, and (e and f) Grade 3 tumors, (a-d, ×200; e-f, ×400; tumor nests-a, c, e; tumor stroma-b, d, f)
Figure 3.
(a-c) H and E stained sections of invasive ductal carcinoma Grade 1, 2, and 3 (×400)
The numerical density of CD68+ TAMs and CD163+ TAMS (M2) was greater in the NDTS than NDT [Table 2].
Table 2.
Tumor-associated macrophages numerical density and its association with Grade of breast carcinoma
| Density (mm2) | Grade 1 (N=25) | Grade 2 (N=25) | Grade 3 (N=25) |
|---|---|---|---|
| CD68 TN | 13 (4) | 20 (15) | 165 (98) |
| CD68 TS | 88 (36) | 178 (76) | 292 (191) |
| CD163 TN | 12 (4) | 15 (7) | 101 (72) |
| CD163 TS | 52 (46) | 130 (84) | 319 (187) |
TS: Tumor stroma, TN: Tumor epithelial nest, N: Number, Values expressed as Mean (Standard Deviation)
TAM expression in breast cancer was associated with the absence of ER and PR. There was no relation to HER2/neu status.
Figure 4 shows one-way ANOVA between various grades of tumor indicating significant differences in terms of CD68 and CD163 densities in TN and TS. This was supported further by significant finding (P < 0.0001) of Bonferroni’s and Turkey’s multiple comparison test. In brief, significantly increased density of CD68 and CD163 was observed in Grade 3 tumor as compared to other two groups. Figure 5 displays CD68 and CD163 densities (mm2) between tumoral (NDT) and stromal (NDS) in different grades of tumor. Results by unpaired t-test indicates increased density in stroma as compared to parenchyma among various grades.
Figure 4.
Comparison between various grades of tumor; (a) CD68 tumor nest (TN) density (mm2), (b) CD68 tumor stroma (TS) density (mm2), (c) CD163 TN density (mm2), and (d) CD163 TS density (mm2).TN(Tumour epithelial Nest), TS(Tumour Stroma). ***P < 0.001 (Significant)
Figure 5.
Comparison of CD68 (a-c) and CD163 (d-f) in density (mm2) between tumor (number density in tumor) and tumor stroma (number density in stroma) in invasive ductal carcinoma. The difference was significant. **= p< 0.001, ***= p< 0.0001
DISCUSSION
TAMs are critical in the regulation of tumor microenvironment in invasive breast carcinoma. There have been studies focusing on the tumor microenvironment with varied results.[4,5]
This study was aimed at quantification of TAMs in IDC using image analyzer software utilizing CD68 and CD163. In this study, increased numerical density of TAM infiltration was associated with advanced tumor grade. High ND of TAMs was found in stroma than intratumoral area.
We found strong correlation of stromal macrophages rather than tumoral macrophages with increasing grades of IDC. In contrast, Gwak et al.[4] in their study found correlation of increased TAMs in all histological locations with intratumoral TAMs being an independent prognostic indicator.
We also discovered that higher TAM expression was associated with absence of ER and PR. Our findings are consistent with the findings of Mahmoud et al., Medrek et al., and Gwak et al. who found that high expression of CD68 or CD163 was linked with negative ER and PR.[2-4,6] We observed no significant connection between HER2/neu status which is also corroborated by Gwak et al.[4] but Mahmoud et al.[2] found correlation with HER2/neu positivity.
In addition, Mahmoud et al. found that macrophage counts are not associated with prognosis in breast cancer and suggested that different subsets of macrophages be investigated to determine their potential involvement.
In the present study, we utilized two immunohistological markers to label the macrophages. CD163, which is a marker for M2 macrophages and the more widely used marker CD68. We have found that CD68 and CD163 labeled macrophages are more in TS than the tumor with significant difference among the grades.
The presence of cytokines and interaction with TME is responsible for division of macrophages into phenotypes. M1 macrophages secrete tumor necrosis factor-a and interleukin (IL-12) which inhibit and lyse tumor cells. Where in M2 macrophages produce IL-1, transforming growth factor-beta, and IL-10 which stimulate tumor development.[8]
IHC for CD68 is the most commonly utilized pan-macrophage marker in tissue sections to delineate both M1 and M2 TAMs.[5] Studies have utilized the overexpression of CD68 as an unfavorable prognostic marker in infiltrating ductal breast carcinoma.
As CD68 stains both M1 and M2 TAMs, we used CD163 a marker for highlighting M2 TAMs in addition.[12]
In the present research, increased ND of CD163 (M2 Macrophages) was significantly related to ER-negative, PR-negative tumors, elevated Ki-67, and an increase in grades. We also found that the numerical density of M2 TAMs was higher in the stroma than intratumoral area. The comparative analysis between the three groups as per the tumor grades indicates increased density of CD68 NDT, CD68 NTS, CD163 NDT density as well as CD163 NDS in Grade 3 tumors, as compared to Grade 1 and 2. Hence, it is assumed that the density of the CD 68 and CD163 expressions increase as per the grade of the tumor. Similarly, it is also observed that CD68 and CD163 densities are more prominent in TS as compared to tumoral area.
In our study, we not only identified the TAM types but also their location within tumors, finding that the ND of TAMs both in tumoral region (NDT) and stroma NDS was related to larger tumors and those with a higher grading.
Discrepancies in quantitative methods such as semiquantitative scoring, counting of overall tumor area, and variability in methodology lead to variable results in studies.[4]
The method used in our study, i.e., morphometric quantitation of TAMs and the use of image analyzer software serves to improve objectivity and is a reproducible method. This would help in standardization of methods to calculate TAMs and compare across studies to improve our understanding of TAMs infiltration in IDC, and thus will help in evaluating the role of TAMs in IDC.
CONCLUSIONS
We used a software for image analysis and computerized system for digital images to undertake quantitative analysis of TAMs on sections stained by CD68 and CD163. This approach standardizes and reproducibly counts the TAMs per unit area. We found significant difference between the number densities of TAMs among grades of invasive breast carcinoma. There were significant differences with ER, PR negativity, and Ki-67. There was no correlation with HER2/neu. CD 68 and 163 densities are more prominent in TS as compared to intratumoral area.
Limitations of the study
The study is limited by the lack of follow-up data and small sample size. Studies of TAMs on a larger sample utilizing this standardized method and follow-up will help in improving our understanding of their role and interaction of TAMs with the tumors.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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