To the Editor,

We noted with interest the study entitled "Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype", published by Ricardo and colleagues [1]. Papers like this one have major importance since retrospective studies analyzing the proportion of cancer stem cells in breast tumor biopsies as prognosis factors are still required. Ricardo et al. also correlate the identification of breast cancer stem phenotypical markers with the molecular subtypes of breast cancer. Therefore, this report tries to address important and relevant questions in breast cancer cell biology. However, this study presents two major flaws. First, the authors performed single CD44 or CD24 staining to identify cells with the CD44+/CD24- cancer stem phenotype. Single immunohistochemistry is not the choice for analyzing the combined expression of two different markers on the same cell but, on the contrary, expression of both receptors needs to been analyzed simultaneously. Double-staining immunohistochemistry for the simultaneous detection of CD44 and CD24 in paraffin embedded sections from breast cancer patients has been developed and validated first by Abraham et al. [2] and subsequently by Mylona et al. [3]. In those reports, the authors quantified the intensity of staining and then the proportion of CD44+/CD24- tumor cells using software-based image analysis in order to avoid bias derived from pathologist inspection. Ricardo and colleagues quote both papers but they followed a different methodology. From our perspective, the double immunofluorescence for CD44 and CD24 performed by the authors in only 10% of the samples does not validate their methodology since it still considered the percentage of cells expressing the receptors rather than the intensity of labeling. On the other hand, they used flow cytometry for the simultaneous analysis of CD44 and CD24 expression in breast cancer cell lines. With this methodology, their results are consistent with those from previous publications [4]. The second imperfection of this study is directly related to the first. Ricardo and colleagues stratified their samples based on the percentage of cells expressing one of the receptors rather than in the number of cells with the CD44+/CD24- phenotype (as reported by Abraham et al. [2] and Mylona et al. [3]). They defined as "CD44 positive" the samples containing 10-100% of tumor cells immunoreactive for CD44 and as "CD24 negative/low" the samples with 0-25% of tumor cells expressing membranal CD24. With this methodology, 411/463 samples (88.6%) were classified as CD24-/low. Thus, nine out of ten samples fitted their description of CD24 "negativity", leaving CD44 "positivity" as the characteristic that is mainly in charge of their whole analysis. Accordingly, the correlation that they found between CD44 expression (not the cancer stem cell phenotype) and the basal subtype (where 94% of the samples were considered CD24-/low) has been previously described in breast cancer cell lines by Charafe-Jauffret and colleagues [5]. Given the clear methodological differences between the present studies from Ricardo and colleagues and those from Abraham et al. [2] and Mylona et al. [3], it is unclear how the authors classified their samples as "CD44+/CD24- <10%" and "CD44+/CD24- >10%" for the further analysis presented in tables 2-3, and figures 2-3. Therefore, we think that the results of this very interesting paper need to be carefully reinterpreted.

References: 1. Ricardo S, Vieira AF, Gerhard R, et al. Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. J Clin Pathol. 2011; doi:10.1136/jcp.2011.090456 2. Abraham BK, Fritz P, McClellan M, Hauptvogel P, Athelogou M, and 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):1154-9. 3. Mylona E, Giannopoulou I, Fasomytakis E, Nomikos A, Magkou C, Bakarakos P, and Nakopoulou L. The clinicopathologic and prognostic significance of CD44+/CD24(-/low) and CD44-/CD24+ tumor cells in invasive breast carcinomas. Hum Pathol. 2008; 39(7):1096-102. 4. Fillmore CM, and Kuperwasser C. Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res. 2008; 10(2):R25. 5. Charafe-Jauffret E, Ginestier C, Monville F, Finetti P, Adelaide J, Cervera N, et al. Gene expression profiling of breast cell lines identifies potential new basal markers. Oncogene. 2006; 25(15):2273-84.

Conflict of Interest:

None declared