Abstract
CD44 + /CD24 − phenotype has been associated with stem cell-like characteristics with enhanced invasive properties, radiation resistance, and with distinct genetic profiles suggesting a correlation to adverse prognosis in western literature. The aim of this study was to study CD44 + /CD24 − phenotype as an adverse prognostic marker in Indian breast cancer patients. N = 61 breast cancer patients included in a tertiary care facility in India were evaluated for receptor studies (estrogen receptor ER, progesterone receptor PR, Herceptin antibody Her2 neu receptor, CD44 & CD24 stem cell markers). CD44 + /CD24 − phenotype was statistically related to adverse factors like estrogen and progesterone receptors non-expression, her 2 neu expression, and triple-negative breast cancer. Of the 39 patients with ER-ve status, 33 (84.6%) were found to have CD44 + /CD24 − phenotype and 82.5% of all the CD 44 + /CD24 − patients were ER negative (p = 0.001). Thirty-four (75.5%) of the PR-ve patients showed the CD44 + /CD24 − phenotype, and of all the CD 44 + /CD24 − patients, 85% of were PR negative (p = 0.006). Thirty-six (75%) of Her-2-Neu + ve were CD44 + /CD24 − . Approximately 90% of the Her 2 Neu patients expressed CD44 + /CD24 − and 76.9% of all the triple-negative patients were found to be CD44 + /CD24 − expression (p = 0.001). CD44 + /CD24 − had a significant association with adverse prognostic factors like stage of disease, hormonal receptor status, and molecular subtypes in Indian breast cancer patients like the Western data.
Keywords: CD44, CD24, Indian breast cancer, Expression, Chemotherapy response
Introduction
Cancer stem cell (CSC) hypothesis was proposed to explore breast cancer heterogeneity which may also contribute to drug resistance and tumor recurrence or metastasis. Breast CSCs are believed to constitute up to 35% of cancer cells in a tumor, whereas these cells constitute only about 1% of stem and progenitor cells present in normal breast [1]. The phenotype of normal human stem/progenitor cells has been described as being aldehyde dehydrogenase (ALDH)high, CD10 + , CD44high, CD24low, or EP-CAM + /MUC-1neg [2]. CD44+/CD24− phenotype in principle has been associated with stem cell-like characteristics with enhanced invasive properties, radiation resistance, and with distinct genetic profiles suggesting a correlation to adverse prognosis [3]. With abundant literature on CD44+/CD24− phenotype expression and its role in treatment and prognosis in Western population, the present study made a similar attempt in breast cancer patients in the Indian subcontinent.
Methodology
This study was done in the Department of General Surgery, King George's Medical University, Lucknow, India, in collaboration with the Department of Pathology, RML Institute of Medical Sciences, Lucknow, India, between August 2013 and July 2014. Tru-cut biopsy/incisional biopsy/post-mastectomy specimens were sent for histopathological examination (HPE) and receptor studies (ER, PR, Her-2-neu, CD44, and CD24 cancer stem cell surface marker expression). All samples were collected before the patient received any sort of systemic treatment.
Each of the immunohistochemical tissue microarray-stained sections were scored using Allred's scoring method [4], which adds scores for the intensity of staining (absent: 0, weak: 1, moderate: 2, and strong: 3) to the percentage of cells stained (none: 0, < 1%: 1, 1–10%: 2, 11–33%: 3, 34–66%: 4, and 67–100%: 5) to yield a “raw” score of 0 or 2–8. Previously validated cutoffs for ER and PR were used (0, 2 = negative, 3–8 = positive) [5, 6]. Her-2-neu status was determined using ASCO/CAP 2018 guidelines [7]. For CD44 and CD24, a score of ≥ 4 was considered positive [6]. Tumors were classified as luminal if they expressed ER or PR and were negative for Her-2-neu. Any tumor with a score of > 5 for Her-2-neu, irrespective of the ER status, was considered a Her-2-neu over-expressing tumor and basal-like tumors were defined as ER, PR, and Her-2-neu negative (triple negative, TNBC) [8].
Statistical analysis was carried out using SPSS 17.0 software. The correlation analyses between the immunophenotypes and the various clinicopathological and biological factors were analyzed. The chi-square test was used for categorical data and p values < 0.05 were considered significant.
Results
N = 61 patients; mean age 47.78 ± 10.04 years, and median 48 years (25–75 years) were analyzed for expression of CD44 and CD24 and its association with tumor factors and hormonal status.
Relation of Phenotype CD44 +/CD24 − with ER, PR, and Her-2-neu Expression
Among ER-ve patients (n = 39), 33 (84.6%) were found to express CD44 + /CD24 − phenotype while 82.5% of all the CD 44 + /CD24 − patients were ER negative (p = 0.001). Similarly, 34/75 (75.6%) PR-ve tumors showed the CD44 + /CD24 − phenotype, and of all the CD 44 + /CD24 − patients, 85% of them were PR-ve (p = 0.006). Thirty-six out of forty-eight (75%) her-2-neu + ve patients were CD44 + /CD24 − while 90% of the CD44 + /CD24 − patients were found to be her-2-neu + (p = 0.003). Majority of the patients belonged to adverse molecular subtypes with high CD44 + /CD24 − expression viz. her-2-neu enriched (92%) and TNBC (76.9%).
Relation of Phenotype CD44 +/CD24 − with TNM Staging
CD44 + /CD24 − expression did not show any significant association with tumor burden (p = 0.449), nodal status (p = 0.054) and presence of metastatic site (p = 0.105), a significant association was noted with overall TNM stage (p = 0.026). However, of all the metastatic breast cancer (n = 9), nearly 90% (n = 8) had CD44 + /CD24 − phenotypic expression (Table 1).
Table 1.
Relation of patient characteristics with CD44 + /CD24 − expression
| CD44 + /CD24 − (% expression) | Others* | P value | |
|---|---|---|---|
| Hormone receptor status (n) | |||
| Estrogen receptor + (22) | 7 (31.8) | 15 | p < 0.0001 |
| Estrogen receptor − (39) | 33 (84.6) | 6 | |
| Progesterone receptor + (16) | 6 (60.0) | 10 | p = 0.006 |
| Progesterone receptor − (45) | 34 (75.6) | 11 | |
| Her2 neu receptor + (48) | 36 (75.0) | 12 | p = 0.003 |
| Her2 neu receptor − (13) | 4 (30.8) | 9 | |
| Molecular subtypes (n) | |||
| Luminal A (9) | 4 (44.4) | 5 | p < 0.001 |
| Luminal B (14) | 3 (21.4) | 11 | |
| Her2 neu enriched (25) | 23 (92.0) | 2 | |
| Triple negative (13) | 10 (76.9) | 3 | |
| Tumor size (n) | p = 0.415 | ||
| T1 (0) | 0 (0) | 0 | |
| T2 (7) | 4 (57.1) | 3 | |
| T3 (44) | 31 (70.5) | 13 | |
| T4 (10) | 5 (50.0) | 5 | |
| Nodal status (n) | p = 0.514 | ||
| N0 (5) | 2 (40.0) | 3 | |
| N1 (44) | 29 (65.9) | 15 | |
| N2 (6) | 5 (83.3) | 1 | |
| N3 (6) | 4 (66.7) | 2 | |
| Metastasis (n) | p = 0.111 | ||
| M0 (52) | 32 (61.5) | 20 | |
| M1 (9) | 8 (88.9) | 1 | |
| Stage (n) | p = 0.026 | ||
| I (0) | 0 (0) | 0 | |
| II (7) | 3 (42.9) | 4 | |
| III (45) | 29 (64.4) | 16 | |
| IV (9) | 8 (88.9) | 1 |
*CD44 + /CD24 + , CD44 − /CD24 + , CD44 − /CD24 −
Discussion
CD44 + /CD24 − has been intensively investigated in Western population with varying results. The only study done on Indian patients for CD44 gene polymorphism by Tulsyan et al. [9] was first to report a taggerSNP-based selection of CD44 gene polymorphisms with breast cancer risk and prognosis in North Indian subjects. In our study, 65.5% patients were found to be expressing CD44 + /CD24 − phenotype which is in agreement with Lin et al. [10] who showed expression in 70% in his study subjects. CD44 + /CD24 − expression was roughly equally distributed in young breast cancer patients (65.2%) and breast cancer patients greater than 40 years (65.8%).
There was no statistical correlation of CD44 + /CD24 − expression with tumor size, nodal status, and metastatic potential; however, it was related to overall stage (p = 0.026). This is in contrast to Lin et al. [10], who showed a significant association between CD44 + /CD24 − and N stage but no association with the patient’s age, tumor size, or TNM stage and distant metastasis. Abraham et al. showed [11] no association between CD44 + /CD24 − and clinical outcome but, in the same study, they did indicate that expression of CD44 + /CD24 − phenotype did correlate with probability of metastasis. Similarly, studies by Perrone et al. [12] and in vitro cell line experiments by Sherdian et al. [13] have explained the role of CD44 + /CD24 − phenotype in probable distant metastasis. The varying results across these studies merit a large well-organized study to come out with definite results.
In Indian breast cancer sub-population, 60% of the patients (39.1% Her-2-neu enriched and 19.6% TNBC) signified poor prognostic subtypes associated with poorer outcomes. Our results showed that majority of ER-negative, PR-negative, and Her-2-neu-positive tumors were CD44 + /CD24 − and vise-verse was true. CD44 + /CD24 − has been associated as a corroborative phenotypic marker for basal-like tumors in various populations of African-American and Hispanic/Latina Women [14]. Lin et al. showed a significantly higher expression of CD44 + /CD24 − in tumors with basal-like features in individuals with local recurrence and metastasis [10]. Ricardo et al. also noticed a similar trend in their study with about 81% of basal-like tumors being CD44 + and 94.5% being negative for CD24 [15]. Ma et al. showed that CD44 + /CD24 − cell population was enriched in TNB tissues and cell lines, with a higher capacity of proliferation, migration, invasion, and tumorigenicity as well as lower adhesion ability [16]. Having said all this, understanding breast cancer biology holds a prime importance with the contrary fact that Kim et al. in the Korean population linked CD44 + /CD24 − as a favorable prognostic subgroup in breast cancer [17]. Thus, CD44 + /CD24 − cell population with cancer stem cell-like properties may play an important role in the aggressive behaviors of TNBC across different ethnic populations.
Conclusion
CD44 + /CD24 − had a significant association with adverse prognostic factors like stage of disease, hormonal receptor status, and molecular subtypes. Large trials with follow-up survival data analysis may provide us with better statistical relationships for evidence-based medicine to practice.
| What is already known on the subject? |
| • CD44 + /CD24 − phenotype is associated with poor prognosis in Western literature |
| Main message |
|
1. CD44 + /CD24 − expression is commonly expressed for advanced stages of carcinoma breast in India as well 2. CD44 + /CD24 − expression is significantly associated with adverse prognostic factors like stage of disease, hormonal receptor status (ER-ve, PR-ve, and Her2neu +), and molecular subtypes (triple negative and Her2neu variant) in Indian breast cancer patients |
| Current research questions |
|
1. Is there any possibility to predict disease free and overall survival on basis of CD44/CD24 expression? 2. Is there any pathological significance (in terms of lymphovascular and perinuclear invasion) of CD44 + /CD24 − expression in breast cancer? 3. Is there any correlation between CD44/CD24 expression and disease recurrence? |
Appendix
Table 2
Table 2.
| Proportion of positive staining score (PS) | Staining intensity score (IS) | ||
|---|---|---|---|
| PS | Range (%) | IS | Type |
| 0 | 0 | 0 | No staining |
| 1 | < 1 | 1 + | Weak positive staining |
| 2 | 1–10 | 2 + | Moderate positive staining |
| 3 | 11–33 | 3 + | Strong positive staining |
| 4 | 34–66 |
Allred score = PS + IS 0–2: negative; 3–8: positive |
|
| 5 | 67–100 | ||
Author Contribution
AkA, S. K., and A. A. S. designed the study. J. L. M., AkA, and K. R. S. were involved in acquisition of data. J. L. M., AkA, K. R. S., and N. H. did the analysis of data. K. G. and AkA drafted the manuscript. A. A. S., M. A., and S. K. critically revised and approved the final draft. AkA, P. A., and A. A. were involved in critical revision and manuscript editing. The manuscript has been read and approved by all the authors, that the requirements for authorship as stated in the instructions of the journal have been met, and that each author believes that the manuscript represents an honest work from this part of the world.
Declarations
Ethics Approval
The study was undertaken as a part of post-graduate thesis after approval from Ethics Committee, King George’s Medical University, Uttar Pradesh, India (Reg no. ECR/262/Inst/UP/2013/RR-19). All the patients were enrolled only after informed written consent post-ethics approval.
Conflict of Interest
Dr. K. G. and Dr. A. A. have shared co-first authorship of this paper since both have worked equally in conceptualizing the study, data analysis, preparation, and revising the manuscript. None of the authors has any conflicts of interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Raouf A, Zhao Y, To K, Stingl J, Delaney A, Barbara M, et al. Transcriptome analysis of the normal human mammary cell commitment and differentiation process. Cell Stem Cell. 2008;3:109–118. doi: 10.1016/j.stem.2008.05.018. [DOI] [PubMed] [Google Scholar]
- 2.Ghebeh H, Sleiman GM, Manogaran PS, Al-Mazrou A, Barhoush E, Al-Mohanna FH, et al. Profiling of normal and malignant breast tissues how CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers. BMC Cancer. 2013;13:289. doi: 10.1186/1471-2407-13-289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Morel AP, Lievre M, Thomas C, Hinkal G, Ansieau S, Puisieux A. Generation of breast cancer stem cells through epithelial-mesenchymal transition. PLoS ONE. 2008;3(8):e2888. doi: 10.1371/journal.pone.0002888. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998;11(2):155–168. [PubMed] [Google Scholar]
- 5.Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol. 1999;17(5):1474–1481. doi: 10.1200/JCO.1999.17.5.1474. [DOI] [PubMed] [Google Scholar]
- 6.Bane A, Viloria-Petit A, Pinnaduwage D, Mulligan AM, O’Malley FP, Andrulis IL. Clinical–pathologic significance of cancer stem cell marker expression in familial breast cancers. Breast Cancer Res Treat. 2013;140:195–205. doi: 10.1007/s10549-013-2591-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Taylor VJ, Barnes PJ, Godwin SC, et al. Assessment of HER2 using the 2018 ASCO/CAP guideline update for invasive breast cancer: a critical look at cases classified as HER2 2+ by immunohistochemistry. Virchows Arch. 2021;479:23–31. doi: 10.1007/s00428-021-03034-4. [DOI] [PubMed] [Google Scholar]
- 8.Tang P, Tse GM. Immunohistochemical surrogates for molecular classification of breast carcinoma: a 2015 update. Arch Pathol Lab Med. 2016;140(8):806–814. doi: 10.5858/arpa.2015-0133-RA. [DOI] [PubMed] [Google Scholar]
- 9.Tulsyan S, Agarwal G, Lal P, Agarwal S, Mittal RD, Mittal B. CD44 gene polymorphisms in breast cancer risk and prognosis: a study in North Indian population. PLoS ONE. 2013;8(8):e71073. doi: 10.1371/journal.pone.0071073. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Lin Y, Zhong Y, Guan H, Zhang X, Sun Q. CD44+/CD24- phenotype contributes to malignant relapse following surgical resection and chemotherapy in patients with invasive ductal carcinoma. J Exp Clin Cancer Res. 2012;31(1):59. doi: 10.1186/1756-9966-31-59. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Abraham BK, Fritz P, McClellan M, Hauptvogel P, Athelogou M, Brauch H. Prevalence of CD44+/CD24−/low cells in breast cancer may not be associated with clinical outcome but may favor distant metastasis. Clin Cancer Res. 2005;11(3):1154–1159. doi: 10.1158/1078-0432.1154.11.3. [DOI] [PubMed] [Google Scholar]
- 12.Perrone G, Gaeta LM, Zagami M, Nasorri F, Coppola R, Borzomati D, et al. In situ identification of CD44+/CD24- cancer cells in primary human breast carcinomas. PLoS ONE. 2012;7(9):e43110. doi: 10.1371/journal.pone.0043110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Sheridan C, Kishimoto H, Fuchs RK, Mehrotra S, Bhat-Nakshatri P, Turner CH, et al. CD44+/CD24- breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res. 2006;8(5):R59. doi: 10.1186/bcr1610. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Wu Y1, Sarkissyan M, Elshimali Y, Vadgama JV (2013) Triple negative breast tumors in African-American and Hispanic/Latina women are high in CD44+, low in CD24+, and have loss of PTEN. PLoS One 8(10):e78259. 10.1371/journal.pone.0078259. eCollection 2013 [DOI] [PMC free article] [PubMed]
- 15.Ricardo S, Vieira AF, Gerhard R, Leitão D, Pinto R, Cameselle-Teijeiro JF, et al. Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. J Clin Pathol. 2011;64(11):937–946. doi: 10.1136/jcp.2011.090456. [DOI] [PubMed] [Google Scholar]
- 16.Ma F, Li H, Wang H, Shi X, Fan Y, Ding X, et al. Enriched CD44(+)/CD24(-) population drives the aggressive phenotypes presented in triple-negative breast cancer (TNBC) Cancer Lett. 2014;353(2):153–159. doi: 10.1016/j.canlet.2014.06.022. [DOI] [PubMed] [Google Scholar]
- 17.Kim HJ, Kim MJ, Ahn SH, Son BH, Kim SB, Ahn JH, et al. Different prognostic significance of CD24 and CD44 expression in breast cancer according to hormone receptor status. Breast. 2011;20(1):78–85. doi: 10.1016/j.breast.2010.08.001. [DOI] [PubMed] [Google Scholar]
- 18.Chen Y, Song J, Jiang Y, Yu C, Ma Z. Predictive value of CD44 and CD24 for prognosis and chemotherapy response in invasive breast ductal carcinoma. Int J Clin Exp Pathol. 2015;8(9):11287–11295. [PMC free article] [PubMed] [Google Scholar]
- 19.Allred DC, Harvey JM, Berardo M. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998;11:155–168. [PubMed] [Google Scholar]
- 20.Dias K, Dvorkin-Gheva A, Hallett RM, et al. Claudin-low breast cancer; clinical & pathological characteristics. PLoS ONE. 2017;12(1):e0168669. doi: 10.1371/journal.pone.0168669. [DOI] [PMC free article] [PubMed] [Google Scholar]