In situ identification of CD44+/CD24- cancer cells in primary human breast carcinomas.

Breast cancer cells with the CD44+/CD24- phenotype have been reported to be tumourigenic due to their enhanced capacity for cancer development and their self-renewal potential. The identification of human tumourigenic breast cancer cells in surgical samples has recently received increased attention due to the implications for prognosis and treatment, although limitations exist in the interpretation of these studies. To better identify the CD44+/CD24- cells in routine surgical specimens, 56 primary breast carcinoma cases were analysed by immunofluorescence and confocal microscopy, and the results were compared using flow cytometry analysis to correlate the amount and distribution of the CD44+/CD24- population with clinicopathological features. Using these methods, we showed that the breast carcinoma cells displayed four distinct sub-populations based on the expression pattern of CD44 and CD24. The CD44+/CD24- cells were found in 91% of breast tumours and constituted an average of 6.12% (range, 0.11%-21.23%) of the tumour. A strong correlation was found between the percentage of CD44+/CD24- cells in primary tumours and distant metastasis development (p = 0.0001); in addition, there was an inverse significant association with ER and PGR status (p = 0.002 and p = 0.001, respectively). No relationship was evident with tumour size (T) and regional lymph node (N) status, differentiation grade, proliferative index or HER2 status. In a multivariate analysis, the percentage of CD44+/CD24- cancer cells was an independent factor related to metastasis development (p = 0.004). Our results indicate that confocal analysis of fluorescence-labelled breast cancer samples obtained at surgery is a reliable method to identify the CD44+/CD24- tumourigenic cell population, allowing for the stratification of breast cancer patients into two groups with substantially different relapse rates on the basis of CD44+/CD24- cell percentage

CD44/CD24 expression in human breast cancer.

<p>A, DAPI; B, DAPI and CD24 (red); C, DAPI and CD44 (green); D, merged images. The composite image (D) shows the heterogeneity of CD44 and CD24 expression. The breast carcinoma cells displayed four distinct sub-populations of cells based on the membrane expression pattern: CD44+/CD24− cells had green membrane staining without membrane CD24 colocalisation; CD44+/CD24+ cells showed a yellow signal along the cell membrane; CD44−/CD24+ cells showed a red signal for CD24 without CD44 staining; and CD44−/CD24− cells were negative for both antibodies. Original magnification, 400×.</p

Analysis of the differences^* between flow cytometry (FC) and immunofluorescence (IF).

*<p>Mann-Whitney Test.</p

Correlation Matrix^* of the clinicopathological and immunohistochemical data.

*<p>Spearman correlation test: the variables were categorised in the analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043110#pone-0043110-t001" target="_blank">Table 1</a>.</p><p>T: tumour size; N: regional lymph nodes; M: distant metastasis; G: differentiation grade.</p

Patient characteristics.

<p>Patient characteristics.</p

CD44/CD24 expression in normal ductal epithelium.

<p>A, DAPI; B, DAPI and CD24 (red); C, DAPI and CD44 (green); D, merged images. Image A shows the double cell layers of breast ductal epithelium composed of basal and luminal cells. Image B shows that CD24 expression is confined to the luminal cell layer, typically in the apical side. Image C shows the typical distribution pattern of the CD44 antigen, which resulted in membrane staining of the basal cell layer and in infiltrating lymphocytes (upper-left corner). D is a merged image of DAPI, CD44 and CD24 images. Original magnification, 400×.</p

Risk of Metastasis (Multivariate Analysis).

<p>Risk of Metastasis (Multivariate Analysis).</p

Analysis of differences between CD44+/CD24− and breast cancer subtypes.

*<p>Kruscal-Wallis Test.</p

Kaplan–Meier survival plots.

<p>Metastasis-free survival in radically resected breast cancer patients according to CD44+/CD24− cells (A), T factor (B), nodal (C) and HER2 (D) status.</p