Aims CD44, CD24 and ALDH1 are the most consistently used biomarkers to identify and characterise the breast cancer stem cell (CSC) phenotype. However, most studies performed until now analysed samples of invasive ductal carcinomas of no special type (IDC-NST). Therefore, prevalence and clinical significance of these CSC markers in breast carcinomas of special histological types (SHT) is largely unknown. For that reason, this study aims to determine the distribution of the breast CD44, CD24 and ALDH1 CSC markers among a series of invasive breast carcinomas of SHT, in comparison with a series of IDC-NST.
Methods 117 invasive SHT breast carcinomas were analysed for the expression of CD44, CD24 and ALDH1, by immuhohistochemistry. The distribution of these CSC markers was evaluated among the distinct histological special types, and the results were compared with a series of 466 IDC-NST.
Results The expression prevalence of the breast CSC markers differed between special types and IDC-NST. Medullary, papillary and tubular carcinomas were enriched in the CSC phenotype CD44+/CD24−/low (80.0%, 100.0% and 100.0%, respectively, vs 45.3% in IDC-NST). Considering the ALDH1 cytoplasmic tumour expression, only medullary and metaplastic carcinomas displayed significant increase in CD44+/CD24-/low/ALDH1+ CSC phenotype frequency (36.4% and 28.6%, respectively, vs 4.8% in IDC-NST).
Conclusions The expression distribution of breast CSC markers is largely dependent on histological type. Interestingly, within the distinct SHT, medullary and metaplastic carcinomas are the two types highly associated with high-grade carcinomas, basal-like and claudin-low molecular subtypes, and to the CSC phenotype CD44+/CD24−/low/ALDH1+.
- Breast Cancer
- Cancer Stem Cells
- Molecular Pathology
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Breast cancer is a heterogeneous disease, comprising various histological types, with distinct clinical presentations and underlying molecular signatures.1 Despite increased knowledge, many patients will develop metastatic disease. One theory that could (at least partially) explain treatment failure is the cancer stem cell (CSC) theory, which hypothesises that cancer may be originated and sustained by a small proportion of cells, displaying the ability to maintain tumour growth by self-renewal and differentiation,2 as well as resistance to chemotherapy3 and radiotherapy.4
Several studies have attempted to demonstrate the presence of breast CSCs (BCSC) based on cell surface markers, being the phenotype CD44+/CD24−/low the most consistently associated with cells displaying stem-like characteristics.5 6 The presence of aldehyde dehydrogenase activity (ALDH) has also been associated with stem cell properties, and tumours expressing ALDH1 are more resistant to platinum-based chemotherapy, more aggressive and associated with worse prognosis.7–10 The significance of combining phenotypes has also been investigated, and it has been shown that ALDH1 expression can further refine CSC CD44+/CD24−/low cell population.11 12 Similar findings have been recently reported by our group, where we demonstrated that CD44+/CD24−/low and ALDH1+ phenotypes are associated with basal-like tumours, both in vitro and in vivo.13
Currently, there is evidence to support the use of CD44 and CD24 cell surface markers in combination with ALDH1 to identify CSC-like cells within invasive ductal breast carcinomas of no special type (IDC-NST). However, regarding special histological types (SHT) that comprise up to 25% of invasive breast carcinomas,14 only a few studies have been conducted exploring the role of CD44 and CD24 in micropapillary carcinomas of the breast (IMPC).15–17 Even in a recent study by Park et al, in which several stem cell-related markers have been tested, only IDC-NST were studied.18 Others used cohorts mainly composed of IDC-NST with only few cases of SHT.12 ,19 Therefore, the frequency of the CSC phenotype in SHT breast carcinomas remains largely unknown.
In the present study, we analysed the immunohistochemical expression distribution of the main established breast CSC markers, namely CD44, CD24 and ALDH1, in a series of SHT of breast carcinomas. In order to determine whether significant differences exist between IDC-NST and SHT, we compared the present results with a large series of IDC-NST previously investigated by our group.13
Material and methods
Breast tumour samples and tissue microarrays (TMA) construction
Formalin-fixed, paraffin-embedded tissues of 117 invasive SHT breast carcinomas were consecutively retrieved from a private laboratory of pathology in Campinas, São Paulo, Brazil, including five lobular classic and 2 pleomorphic, 16 tubular, 26 mucinous, six IMPC, eight invasive papillary, four typical and 16 atypical medullary, 10 metaplastic and 24 apocrine carcinomas. All cases were reviewed on haematoxylin and eosin-stained (H&E) sections by two pathologists (RG and FS), and classified according to the WHO Classification of Tumours of the Breast.20
Representative tumour areas were selected on H&E sections and marked for construction of TMAs with 2 mm in diameter with two tissue cores obtained from each specimen. Detailed conditions for TMA construction can be found on online supplementary materials.
The expression of commonly used biomarkers was evaluated, namely the hormone receptors ER and PgR, the proliferation marker Ki67, the tyrosine kinase receptors HER2 and endothelial growth factor receptor (EGFR), CK5 and also P-cadherin. Immunohistochemistry was performed as previously reported.13 Detailed conditions for each antibody can be found in online supplementary material table I. In order to translate the immunohistochemical findings to the molecular subtype, we considered luminal tumours when there was expression of ER, HER2 overexpressing (HER2-OE) when the tumour demonstrated HER2 expression without ER or PgR expression, and triple-negative tumours when the three markers were absent. The expression of the breast cancer biomarkers ER, PgR, HER2, EGFR, CK5 and P-cadherin was evaluated according to the grading systems already described in a previous work by our group.21
To compare the present results with the ones previously reported in a large series of IDC-NST, the expression of CD44, CD24 and ALDH1 was evaluated and scored as published.13 Detailed methodology used for CD44 and CD24 is described in online supplementary materials. ALDH1 expression was classified as positive, when more than 1% of tumour cells showed clear cytoplasmic positivity.7 ,10 Stromal expression of ALDH1 was also classified as previously described.22 In selected cases, immunohistochemical evaluation was performed on the whole tissue section.
Associations between the different parameters were assessed using the χ2 test or Fisher's exact test when appropriate. Statistical analyses were conducted using SPSS statistics V.20.0 software and a two-tailed significance level of 5% was considered as statistically significant. Estimated risk differences (RD) for the phenotypes CD44+/CD24−/low and CD44+/CD24−/low/ALDH1+ are also presented with 95% CI values.
CD44 And CD24 expression within special types and associations with clinicopathological parameters
The expression of CD44 was analyzed in 87 cases, with 78.2% (68/87) demonstrating a clear positive membrane staining, thus indicating an increase over the prevalence of this marker in the IDC-NST comparison series (p<0.001) (see online supplementary material table II). Regarding specific types, CD44 was differentially expressed in both medullary and tubular carcinomas, which displayed an enrichment in this marker when compared with IDC-NST (both associations with p<0.001). CD44 expression in this series was not associated with any of the clinicopathological parameters evaluated (see online supplementary material table III) (figures 1 and 2).
Concerning CD24 expression, there were no differences in this series expression when compared with IDC-NST, being 18.9% (17/90) of positive cases. Expression was also not different between subtypes. However, when each subtype was compared with IDC-NST, invasive apocrine and papillary carcinomas displayed an increased expression of this marker (42.9% and 100% vs 11.4% in IDC-NST, with p=0.004 and p<0.001, respectively). Furthermore, CD24 expression was associated with high-grade tumours, with 58.8% (10/17) of the positive cases classified as grade III carcinomas (p=0.011), as well as with ER negativity (p=0.009) and HER2 overexpression (p=0.005). These results could be translated into differences between the molecular subtypes, with only 9.8% (6/61) of luminal cases classified as CD24-positive (p=0.004) (see online supplementary material table IV).
ALDH1 expression within special types and association with clinicopathological parameters
Concerning ALDH1 expression, both the cytoplasmic and stromal expression of ALDH1 were evaluated, with 13.3% (8/60) considered positive for cytoplasmatic expression in tumour cells and 84.6% (21/25) classified as positive for stromal expression (see online supplementary material table III).
Specifically regarding cytoplasmic expression, ALDH1 was differently expressed between the several types; when compared with the IDC-NST series, both medullary and papillary carcinomas displayed increased prevalence of ALDH1 cytoplasmic expression (41.7% and 100%, respectively, vs 7.1% in IDC-NST, both p<0.001) (figures 1 and 2). Medullary and papillary carcinomas also displayed increased prevalence of ALDH1 stromal expression (both with all cases expressing ALDH1 vs 36.7% in IDC-NST, p<0.001 and p=0.019, respectively); all metaplastic carcinomas expressed ALDH1 in the stroma (p=0.019) (see online supplementary material table III). ALDH1 cytoplasmic expression was significantly associated with high-grade tumours (p=0.025), ER negativity (p=0.010), PgR negativity (p=0.022) and basal markers expression, namely EGFR (p=0.014), CK5 (p=0.013) and P-cadherin (p<0.016). ALDH1 expression was also associated with triple-negative tumours (p=0.010) (see online supplementary material table IV).
CSC phenotypes (CD44+/CD24−/low and CD44+/CD24−/low/ALDH1+) and association with clinicopathological parameters
To further explore the association between tumour characteristics and the CSC phenotypes, we decided to consider a tumour with CSC phenotype when the frequency of CD44+/CD24−/low cells was higher than 10%, as previously described.14 ,23 Additionally, similar to the approach by Raza Ali et al,12 we defined a putative CSC phenotype by adding ALDH1 cytoplasmic expression, thus defining a CD44+/CD24−/low/ALDH1+ phenotype.
The CSC phenotype CD44+/CD24−/low was observed in 65.5% (57/87) of the cases, with only 45.3% (209/461) of IDC-NST cases considered positive (p<0.001, RD: −0.20 (−0.30, −0.09)). The CD44+/CD24−/low phenotype was also differently prevalent among subtypes (p=0.002), with tubular carcinomas displaying an increased prevalence (p<0.001, RD: −0.55 (−0.59, −0.33)).), with all cases (n=14) positive for this characteristic (after immuhistochemical confirmation on whole tissue sections). Medullary and papillary carcinomas also displayed an increased prevalence of this CSC phenotype (80% and 100%, with p=0.008 and p=0.043, RD: −0.35 (−0.48, −0.09) and RD: −0.55 (−0.59, −0.06), respectively) (table 1).
For the CSC phenotype CD44+CD24−/lowALDH1+, its prevalence was higher in SHT (11.5%, 6/52) than in IDC-NST (4.8%, 22/437) (p=0.043, RD: −0.20 (−0.30, −0.09)), and significantly differed between the several SHT (p=0.042). The two types that displayed an increased prevalence of this phenotype were the medullary carcinomas, with 36.4% (4/11) of positive cases, and the metaplastic carcinomas, with 28.6% (2/7) of positive cases (p=0.002 and p=0.046, with RD: −0.32 (−0.60, −0.10) and RD: −0.24 (−0.59, −0.03), respectively).
When these phenotypes were correlated with pathological variables and markers, the phenotype CD44+/CD24−/low was only significantly associated with negative HER2 status (p=0.018) and high-grade tumours (p=0.005). In case of the CD44+/CD24−/low/ALDH1+ phenotype, it was correlated with ER and PgR negativity (p=0.008 and p=0.009, respectively) and with the presence of basal markers, namely EGFR (p=0.022) and CK5 (p=0.001). The CD44+/CD24−/low/ALDH1+ phenotype was further associated with triple-negative tumours in comparison with the luminal ones (p=0.008) (see online supplementary materials table V).
The present study was designed to evaluate the heterogeneity found in breast CSC markers expression within the different SHT. To our knowledge, only two studies specifically investigating the expression of CSC markers and some SHT have been published. In one of these, only the expression of CD44 was evaluated in IMPC and compared with tubular carcinomas;15 with the other only reporting the comparison of the immunophenotype CD44+/CD24−/low between IMPC and IDC-NST.16 Therefore, our study is the first to address the question of CD44, CD24 and ALDH1 CSC markers expression prevalence, their association with pathological features and with distinct biomarkers in a cohort of different SHT. Furthermore, it compares each SHT represented with our previously published data on a large cohort of IDC-NST, to explore a possible enrichment of these markers in specific SHT.
CD44 was more commonly expressed in both tubular and medullary carcinomas. Although tubular carcinomas are a low-grade, well-differentiated SHT, similar findings regarding high prevalence of CD44+ cells have already been described,15 CD44+ cells have also been associated with a mesenchymal stem cell-like profile, and also inversely associated with lymph node metastasis, as shown by Giatromanolaki et al24 and ourselves.13 Interestingly, both tubular and medullary carcinomas are SHT of breast cancer rarely associated with lymph node metastasis when compared with grade-adjusted IDC-NST,25 ,26 probably due to low metastatic potential in case of tubular carcinomas and to haematogenous metastatic route in case of medullary carcinomas.28
Concerning CD24 expression, in comparison with IDC-NST, only invasive apocrine and papillary carcinomas displayed a significantly increased expression of this marker, contrary to a previous report of an increased prevalence of CD24 expression in IMPC.16 When considering clinicopathological associations, we found an association with negativity for ER and HER2 expression, as well as with higher prevalence of expression in grade III tumours. Although these findings could be explained by the large representation in our series of CD24− tubular carcinomas, some authors have reported similar associations, namely between CD24 expression and carcinomas with higher grade28 and unfavourable prognosis .23 ,29 Still, the significance of its expression remains controversial13 ,16 with some studies associating it with worse prognosis23 unlike some others.6
When investigating ALDH1 in special subtypes, only papillary and medullary carcinomas demonstrated increased prevalence of expression of this marker over IDC-NST. ALDH1 cytoplasmic expression was also associated with high-grade, triple-negative tumours and the expression of basal-like markers, similar to that previously described by our group and others.12 ,13
When the CSC immunophenotypes were assessed, we found a similar prevalence of CD44+/CD24−/low tumour cells in the SHT series in comparison with the one observed in IDC-NST series.16 ,19 ,23 However, tubular, papillary and medullary carcinomas were associated with a higher prevalence of CD44+/CD24−/low CSC phenotype. Other authors have also briefly reported this association with medullary carcinomas.19 Nevertheless, we could not confirm the reported enrichment of the CD44+/CD24−/low phenotype in the IMPC16 special type, nor could we find any association with CK5, CK14 and EGFR expression, or with ER negativity, as reported in cohorts predominantly composed of IDC-NST.12 ,19
By contrast, the CSC phenotype CD44+CD24−/lowALDH1+, previously associated with a worse patient prognosis in a subgroup of ER-negative tumours,12 was associated with ER and PgR negativity, triple-negative tumours, and also with the presence of basal markers, namely EGFR and CK5.
More interestingly, only medullary and metaplastic carcinomas demonstrated this CSC phenotype CD44+CD24−/lowALDH1+, with a significant increase of its prevalence over IDC-NST. Therefore, in our SHT series, the use of the CSC phenotype CD44+/CD24−/low/ALDH1+ specifically identified the two breast cancer SHT consistently associated with basal-like molecular subtypes, as previously demonstrated in triple-negative IDC-NST tumours.12 ,13 ,19
One potential limitation of our study is that our series contained several samples, without any adjustment in the proportion of each SHT for known prevalence. Consequently, special care should be taken when interpreting the clinicopathological associations. However, since each SHT was compared individually with the IDC-NST series, we consider that the main objective of this study was not affected by the sampling methodology. Another possible limitation was the use of a historical control series; however, potential biases were limited by the use of the exact same methodology for performing IHC, the same scoring system, and even the same pathologists interpreting the samples in the two studies.
In summary, we demonstrated that the prevalence of CSC markers is heterogeneous among breast cancer SHT, thus providing further evidence that the several SHT are distinguished entities from IDC-NST also in what concerns CSC markers expression. Moreover, we provide evidence that, by using the CD44+/CD24−/low/ALDH1+ CSC phenotype, we can identify the SHT characteristically associated with basal-like and claudin-low molecular subtypes.
Prevalence of CSC markers is heterogeneous among breast cancer special histological types and differs from IDC-NST;
Although low-grade and luminal type, tubular carcinomas display the phenotype CD44+CD24−/low;
Metaplastic and medullary carcinomas, which are high-grade and basal-like, are the two special types, enriched in the CD44+/CD24−/low/ALDH1+ CSC phenotype.
What this paper adds
▸ To our knowledge, a few authors have only briefly addressed the issue of CSC markers expression in special histological types of breast cancer. Thus, this is the first report evaluating the prevalence of CD44, CD24 and ALDH1 CSC markers in a cohort of distinct breast cancer histological special types. Furthermore, by comparing the results obtained with our previously published results in a large series of IDC-NST, we demonstrated that different special types show a different enrichment in these markers when compared with IDC-NST. Additionally, we found that both, medullary and metaplastic carcinomas, which are the two special types associated with high-grade carcinomas, as well as with the basal-like and claudin-low molecular subtypes, are the ones highly enriched in the CSC phenotype “CD44+/CD24-/low/ALDH1+ CSC phenotype”.
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Contributors FB: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; PC, RG, CA, JP: collection and/or assembly of data and data analysis and interpretation; MG: collection and/or assembly of data; FB, RG, FS: data analysis and interpretation; FS: conception and design, data analysis and interpretation, manuscript writing and final approval of manuscript.
Funding This work was supported by Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP, grant number 2010/00815-6), CAPES- Ministry of Education, Brazil (César A Alvarenga: PhD grant 3420-09-04) and Fundação para a Ciência e Tecnologia, Portugal (Madalena Gomes: PIC/IC/83264/2007). IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by FCT. This work was also supported by the scientific project number13531, funded by FEDER: Sistema de Incentivos à Investigação e Desenvolvimento Tecnológico, Programa Operacional de Factores de Competitividade, for Renê Gerhard.
Competing interests None.
Ethics approval This study was conducted under the national regulative law for the handling of biological specimens from tumour banks, being the samples exclusively available for research purposes in retrospective studies.
Provenance and peer review Not commissioned; externally peer reviewed.
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