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Abstract
Introduction: Cancer stem cells (CSC) within the tumors play a central role in tumorigenesis. It is, thus, of utmost importance to identify these cells to develop effective cancer therapy. Triple-Negative Breast Cancer (TNBC) is an aggressive molecular subtype of breast cancer associated with poor patient outcomes. The role of CD44 immunohistochemistry (IHC) as a putative CSC in breast carcinomas, particularly of the TNBC-subtype is ambiguous, with equivocal results. Aims and Objectives: The present study aims to assess the role of CSC in breast carcinoma by immunohistochemical analysis of CD44 expression in TNBC. The association of TNBC expressing CSC with histological grade as well as with angiogenesis (using CD34 IHC) has been studied. Materials and Methods: Biopsy samples from 58 patients with infiltrating ductal carcinoma, NST were studied. The histology of the tumor was sub-classified into grades 1–3. Based on immunohistochemical analysis (ER, PR, HER2/Neu), the cases were divided into TNBC and NTNBC groups. The tissue sections were also subjected to analysis for CD44 to identify the CSC-phenotype and CD34 to evaluate angiogenesis, to determine the microvascular density (MVD). Results: Out of the 58 cases in the study, 28 were TNBC and 30 were NTNBC. CSC phenotype (CD44 positive) was expressed significantly higher in the TNBC (78%) versus the NTNBC (53%) (p-value 0.043). The MVD estimated using CD34 IHC was lower in the TNBC group in our study, though the difference was not statistically significant. A larger proportion of cases in TNBC showed a higher histological grade (35%) in comparison to NTNBC (27%). However, statistically, it was not significant. Conclusion: Our study demonstrated that CD44 as a CSC marker is seen significantly more in the TNBC category of invasive ductal carcinomas. Further large-scale studies, to confirm these findings, will be of potential therapeutic and prognostic value.
Keywords: Breast cancer, cancer stem cells, CD44, immunohistochemistry, TNBC
How to cite this article: Introduction 
Breast cancer is the most commonly diagnosed cancer in females worldwide as well as the leading cause of cancer-related deaths.[1] Diversity in breast cancer is known, with numerous histological and molecular subtypes being identified. Subtyping of breast cancer according to molecular and genetic subtypes has an impact on the prognostication of disease and selection of treatment modalities. Triple-negative breast cancer (TNBC) is known as a type of breast cancer in which there is a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and Human Epidermal Growth Factor Receptor 2 (HER2). Targeted therapy is available for ER or PR positive as well as HER2 positive categories, but no standard treatment option is available for TNBC.[2] Owing to a markedly poorer prognosis in this subset and lack of specific targeted therapy, much research is going on in this category of breast cancer.
Many studies have provided insights into the pathogenesis of neoplasms, with the identification of cancer stem cell (CSC) niches in tumors playing a central role in tumorigenesis. It is, thus, of paramount importance that these specific cells within the tumor are better-characterized to develop effective anticancer therapy. Various techniques have been developed to identify CSC. Immunohistochemical staining for stem cell markers such as CD44 allows excellent visualization of such cells.
While the literature is replete with numerous studies evaluating the role of CD44 immunohistochemistry (IHC) as a stem cell marker in various tumors, there is a paucity of literature evaluating its potential role in breast carcinoma, especially the TNBC, which are known to have a poor prognosis and survival.[3–18]
The present study aimed to gain better insight into the role of CSC in breast carcinoma by immunohistochemical analysis of CD44 expression in TNBC. The association of TNBC expressing CSC with histological grade as well as with angiogenesis (using CD34 IHC) has been studied.
Materials and Methods This is a retrospective descriptive study including biopsy samples from 58 female patients with carcinoma breast, of no special type (NST). Histological types of invasive breast carcinoma apart from NST, like mucinous, lobular, and so on. were excluded from the study. Patients with prior neoadjuvant chemotherapy and/or radiation therapy and male patients with carcinoma breast were also excluded.
Detailed clinical history, examination findings, and radiological data were recorded from the existing data records. The paraffin-embedded tissues were sectioned and stained with routine Hematoxylin and Eosin using the standard protocol. Microscopic analysis of each specimen was done for confirmation of diagnosis as well as grading using the standard Elston/Nottingham modification of the Bloom Richardson grading system.[19]
Immunohistochemistry
Cases diagnosed as infiltrating ductal breast carcinoma, NOS were subjected to immunohistochemical analysis for ER, PR, HER 2 neu (hormonal profile), CD44 (stem cell marker), and CD34 (for angiogenesis). About 4–5-μm thin sections were deparaffinized and rehydrated. To block the endogenous peroxide activity, the sections were placed in 3% hydrogen peroxide diluted in nine parts of methanol for 30 min. Target antigen retrieval was achieved using citrate buffer (pH 6.0) in the microwave. The sections were incubated overnight in a moist chamber with primary antibodies ER, PR, HER2/Neu, CD44, and CD34. The next day after bringing them to room temperature and washing, the secondary antibody (biotinylated anti-mouse polyclonal by Dako) was applied and the sections were re-incubated for 1 hour. Chromogen (Diaminobenzidine) was added for 1–3 min and checked under a microscope. Counterstaining was done with hematoxylin for 1 min.
Evaluation of Immunohistochemical expression
ER and PR:
The samples were considered positive for ER or PR if ≥1% of tumor cell nuclei were immunoreactive [Figure 1].[20]
Figure 1: (a) Nuclear positivity seen for ER (400x, IHC). (b) ER negative (200 x, IHC). (c) Nuclear positivity seen for PR (400 x, IHC). (d) PR negative (200 x, IHC). (e) Membranous positivity seen for HER2 (400 x, IHC). (f) HER2 negative (200 x, IHC)HER2/Neu
Reporting Her2neu was done as per the standard criteria.[21]
Positive:
IHC 3+ (strongly positive) tumor displays complete, intense, circumferential, membranous staining in >10% of tumor cells [Figure 1].
Equivocal:
IHC 2+: weak to moderate complete membrane staining observed in >10% of invasive tumor cells.
Negative:
IHC 1+: incomplete faint membrane staining and within >10% of invasive tumor cells.
IHC 0: no staining observed or incomplete, faint/barely perceptible membrane staining within ≤10% of invasive tumor cells.
Based on ER, PR, and HER2Neu expression, breast cancer was categorized into TNBC (ER−, PR−, and Her2−) and NTNBC (ER + PR + HER2−, ER + PR − HER2−, ER − PR − HER2+, ER + PR + HER2+, ER + PR − HER2+, ER-PR + HER2- and ER-PR + HER2+) phenotypes.
CD 44
Assessment of positivity or negativity for CD44 was performed semi-quantitatively from immunohistochemically stained sections of tumor samples from patients. Tumor cells were localized using 10x objective and the level of positivity was graded for CD44 staining as 0 (negative), 1 (mild), 2 (moderate), and 3 (strongly positive) [Figure 2]. Positive staining for CD44 was defined as minimum 2+ circumferential membranous staining in atleast 10% of the tumor cells.[13],[22] Only membranous staining in tumor cells is scored and included in our study; stromal or non-membranous staining is not included.
Figure 2: CD44 immunostain (400 x, IHC). (a) Negative (b) 1+ (c) 2+ (d) 3+Estimation of microvascular density (MVD) using CD34.[23]
The vascular endothelium was highlighted using CD34 immunostaining. Angiogenesis was measured by counting the CD34-labelled microvessels. Any endothelial cell cluster with or without lumen, clearly separated from adjacent microvessels, was counted as a distinct microvessel. Larger vessels, having a muscular wall, were excluded from this count [Figure 3].
Figure 3: (a-c) Infiltrating ductal carcinoma, NST (Hematoxylin and Eosin stain; 100 x, 200 x and 400 x respectively). (d) CD34 immunostain highlights the microvessels (IHC, 200 X)Under low power objective, (100 x magnification) the areas with the highest density of vascular staining were identified as hotspots. Under higher magnification (400 x), the microvessel counting was done in five, non-repetitive, hotspot areas. Average MVD was calculated by dividing by the field area.
Data analysis
The results were tabulated and statistical analysis was done using Student's t-test to look for a correlation among the histological parameters and expression of ER, PR, HER2/Neu with CD44, and angiogenesis. A P value <0.05 was considered significant. SPSS (IBM SPSS Statistics for Windows, Version 25 Armonk, NY: IBM Corp.) was used for analysis.
Results Out of the total 58 breast carcinoma cases in this study, 40 cases (69%) were histological grade 2 and 18 (31%) belonged to grade 3. Based on hormonal profile (ER, PR, HER2), 28 cases were TNBC and 30 were NTNBC. These cases were taken in a comparable number (28 vs. 30) in both groups for statistical purposes. CSC marker CD44 was expressed in 22/28 (78%) cases of Triple-negative receptor type of breast cancers (TNBC) and 16/30 (53%) in non-Triple negative receptor type of breast cancers (NTNBC). (p-value 0.043) [Table 1].
The MVD estimated using CD34 IHC was lower in the TNBC group (9.73/mm2 vs. 11.15/mm2) in contrast to NTNBC in our study, though the difference was not statistically significant [Table 2].
Though the majority of the TNBC as well as NTNBC cases belonged to grade 2 (64% and 73%, respectively), a larger proportion of cases in TNBC showed higher histological grade (35% in comparison to 27%). However, statistically, it was not significant [Table 3].
Discussion TNBC is an immunohistochemically distinct group of breast cancers, which is defined by a negative expression of ER, PR, and HER2/Neu. Compared with other forms of breast cancer, TNBC is associated with a younger age at diagnosis, advanced stage, increased risk of, and poorer outcome.[2] These are not responsive to conventional receptor-target therapies also. Owing to the prognostic and therapeutic impact, TNBC has acquired much scientific interest in recent years.
The role of CSC was first described in breast cancer by Al-hajj and his co-workers with the cell-membrane markers CD44 and CD24.[3] The concept emphasizes the presence of a subpopulation of cells within tumors that propel tumorigenesis. CSC are also known as tumor-initiating cells (TIC), or cancer metastasis-initiating cells (CMICs). CSC are especially shown to exhibit unique growth abilities including self-renewal and differentiation potential. Identification of CSCs and establishing their role in the pathogenesis of neoplasm may play a pivotal role in developing CSC targeting treatment in the future.
CD44, CD133, and ALDH1 are the most widely searched cellular markers for the detection of CSC.[12] CD44 is a transmembrane glycoprotein that is involved in cell–cell interactions, cell adhesion, and migration. Besides, it promotes EGFR-mediated pathways, consequently leading to tumor cell growth, tumor cell migration, and chemotherapy resistance in solid cancers.[3] Literature shows CD44 to be a critical player in numerous tumors including colon cancer, prostate cancer, as well as squamous cell carcinoma of the head and neck region.[24],[25] In breast cancer, few studies are available evaluating the compelling role of CD44 immunohistochemistry.[26] Accumulating evidence suggests that the breast cancer cells with CD44+ subpopulation express higher levels of preinvasive genes and have greater invasive potential.[13]
The present study demonstrated that CD44 expression is significantly associated with TNBC phenotype (p-value <0.05). [Table 4] summarizes the CD44 expression by IHC, reported in various studies. Similar to the present study, Idowu et al. have concluded that the patients with TNBC had a significantly higher frequency of breast CSC phenotype (CD44+/CD24-/low).[13] Furthermore, the correlation of CD44+ immune-expression with the aggressive clinical behavior of TNBC has also been highlighted in their paper. Paula et al. in 2016 evaluated triple immunohistochemistry (CD44, ALDH1, and Ki67) in their work on 16 normal, 54 non-malignant, and 155 malignant breast tissues.[27] They established that the mean percentages of cells with CD44+, ALDH+, and Ki67-phenotype increased significantly from non-malignant lesions to high-grade ductal carcinomas in situ, however, decreased in invasive ductal carcinomas. Nevertheless, this phenotype was associated with distant metastasis in patients with ductal carcinomas of the breast.[28] Some studies have also concluded the CD44+/CD24-status to be associated with basal-like molecular subtype[17] Wang et al. concluded that CD44/CD24 status evaluated by double-staining IHC constitutes an independent prognostic factor for TNBC.[29] Klingbeil et al., in their aCGH analysis on CD44 expression, concluded that it is particularly enriched in TNBC, which has the worst clinical prognosis and outcome.[30]
Table 4: Comparative table of CD44 staining results in various studies[6],[8],[9],[10],[13],[26],[27],[33],[34],[35],[36],[37],[38],[39],[40],[41]The present study showed a greater proportion of TNBC to be demonstrating higher histological grade, and conceivably an extrapolatable poorer prognosis; but the correlation is statistically insignificant (p-value >0.05). Literature shows that TNBC is a heterogeneous group, and most TNBCs are high-grade invasive ductal carcinomas, but there is also a subset of TNBCs comprising histologically low-grade lesions whose clinical course and molecular features differ from the high-grade TNBC.[2],[8],[27] A comprehensive meta-analysis conducted by Wang et al. including 16 studies on breast cancer patients also concluded that no association was observed between CD44 and histological grade complying with our findings.[15]
Angiogenesis plays a central role in the neoplastic process, promoting tumor growth and metastasis. Assessment of tumor MVD as a marker of angiogenesis has been abundantly researched and the development of agents that inhibit tumor angiogenesis has been an active area of investigation. Observational studies have demonstrated that MVD is a prognostic factor in invasive breast cancer, whereas others reached the opposite conclusion. Few studies have reflected that MVD in both basal-like and TNBC is significantly higher than in non-basal-like and non-TNBC.[31] In our study, the MVD in two comparison groups of breast carcinoma revealed an increase in MVD in the NTNBC group, which was, however, statistically insignificant (p-value >0.05). Our study has excluded post- chemo and radiotherapy specimens, because of which the MVD has been analyzed on trucut cores. As there is a marked heterogeneity in micro-vascularization in any tumor, perhaps a careful search for possible hotspots in larger resection specimens can provide more representative results.
Previous studies have suggested that CD44 expression is related to tumor invasion, metastasis, and tumor progression, but there is a lack of clarity in terms of its prognostic value due to conflicting results.[7],[9],[12],[15] On the one hand, studies have found that CD44+ breast cell lines have invasive and metastatic properties, correlating with poorer prognosis.[14],[16],[32] On the other hand, it has also been documented that loss of CD44 promoted metastasis, by some researchers.[30] Consequently, there is a need for larger sample studies to answer the question of whether CD44 is a true prognostic marker of patients' outcomes or a predictive marker of sensitivity to therapy. Research insinuates that the use of monoclonal antibodies against CD44 may be a landmark therapeutic achievement for advanced malignancies.[3] A lacuna in the present study is the lack of follow-up of the cases in terms of treatment and clinical outcome, which might have contributed to the prognostic value. Furthermore, the use of additional stem cell markers, in larger studies, might further add to their potential role in breast cancers.[41]
Conclusions The present study deduces that CD44 + phenotype is expressed significantly more in triple-negative receptor type of breast cancers. However, no statistically significant correlation was found with the histological grade or angiogenesis in breast carcinoma. With the potential therapeutic implication of CD44 (cancer stem cell) in TNBC, this study adds to the limited existing literature available and can lay the foundation for further larger studies to substantiate these findings.
Ethics statement by all authors
The study was granted an exemption by the institutional research ethics committee of Maulana Azad Medical College on the grounds of research involving the collection or study of existing data and diagnostic specimens vide (F.1/IEC/MAMC/83/01/2021/No. 330).
List of abbreviations (In alphabetical order)
CMICS: Cancer Metastasis-Initiating CellsCSC: Cancer Stem CellER: Estrogen ReceptorHER2: Human Epidermal Growth Factor Receptor 2IHC: ImmunohistochemistryMVD: Microvascular DensityNST: No Special TypePR: Progesterone ReceptorTIC: Tumor-Initiating CellsTNBC: Triple-Negative Breast Cancer.Financial support and sponsorship
This study received financial support from institute under intramural research project.
Conflicts of interest
There are no conflicts of interest.
References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_437_21
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