Cytoplasmic CD24 Expression in Colorectal Cancer Independently Correlates with Shortened Patient Survival

Materials and Methods

Cell lines. The human colon adenocarcinoma cell line HT29 was obtained from American Type Culture Collection (Rockville, MD). The human colon adenocarcinoma cell line CX-2 was from the German Cancer Research Center (Heidelberg, Germany). Cells were cultured in RPMI supplemented with 10% fetal bovine serum.

Patients. One hundred forty-seven consecutive cases (age, 17 to 87 years; median, 65) who were diagnosed for colorectal cancer at the Institute of Pathology, Charité University Hospital, between 1996 and 1998 were enclosed in this study. Only patients with primary colorectal adenocarcinomas without neoadjuvant radiochemotherapy and no other known malignancies at the time of diagnosis and during follow-up were included. A small number of cases had to be excluded due to limited tissue availability. Histologic diagnosis was established on standard H&E-stained sections according to the guidelines of the WHO. The vast majority of patients with nodal positive colon cancer and patients with either nodal positive or locally advanced (pT₃/pT₄) rectal cancer received adjuvant chemotherapy with 5-fluorouracil and levamisole or 5-fluorouracil alone according to the guidelines of the German Cancer Society from 1994 (18). Clinical follow-up data were available for all patients. Forty-one (27.9%) patients died during follow-up with a median survival of 17 months. The median follow-up time of patients still alive at the end point of analysis was 53 months. Clinicopathologic variables are summarized in Table 1.

Immunofluorescence. Immunofluorescence staining was done according to standard procedures. Briefly, subconfluent cells grown on Labtek chamber slides (Nunc, Wiesbaden, Germany) were fixed in methanol for 10 minutes at −20°C. Slides were incubated with primary CD24 antibody (clone 32D12, Dianova, Hamburg, Germany) diluted 1:100 in PBS/1% bovine serum albumin for 90 minutes at 21°C, followed by incubation with a Cy3-conjugated anti-mouse antibody (Dianova) diluted 1:200 in PBS/1% bovine serum albumin. Confocal laser scanning microscopy was done using a Leica confocal microscope.

Immunohistochemistry. Formalin-fixed paraffin-embedded tissue was freshly cut (4 μm). The sections were mounted on superfrost slides (Menzel Gläser, Braunschweig, Germany), dewaxed with xylene, and gradually hydrated. Antigen retrieval was achieved by pressure cooking in 0.01 mol/L citrate buffer for 5 minutes. The primary CD24 antibody (Ab-2, clone 24C02/SN3b, Neomarkers, Fremont, CA) was diluted 1:100 using a background reducing dilution buffer (DAKO, Hamburg, Germany) and was incubated at room temperature for 1 hour. Detection took place by the conventional labeled streptavidin-biotin method (LSAB kit, DAKO) with alkaline phosphatase as the reporting enzyme according to the instructions of the manufacturer. Fast Red (Sigma-Aldrich, Munich, Germany) served as chromogen. Afterwards the slides were briefly counterstained with hematoxylin and aqueously mounted.

Immunostaining of tissue slides was independently evaluated by three clinical pathologists who were unaware of patient outcome. As CD24 showed a cytoplasmic and membranous staining, we scored both staining qualities as either negative or weakly, moderately, or strongly positive.

Statistical analysis. The data were compiled with the software package SPSS, version 12.0. Fisher's exact test, χ² test, and χ² test for trends were used to assess the statistical significance of associations between expression of CD24 and clinicopathologic variables. Univariate survival analysis was done according to Kaplan-Meier; differences in survival curves were assessed with the log-rank test. Multivariate survival analysis was done on all variables that were found to be significant on univariate analysis using Cox's regression model, having checked that no violation of the proportional hazards assumption became apparent in a visual evaluation of log(−log(survival)) plots. P < 0.05 was considered significant. All statistics were accredited by the head biostatistician of the Tumor Center, Charité University Hospital (J.B.).

Results

CD24 immunofluorescence. The cell line CX-2 showed a weak and only focal dot-like membranous CD24 immunoreactivity (Fig. 1A). In contrast, HT29 cells exhibited a strong homogeneous cytoplasmic and focal membranous immunostaining (Fig. 1B).

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Fig. 1.

CD24 immunofluorescence. A, CD24 expression is virtually absent in the colon cancer cell line CX-2. Note strong exclusively membranous expression in the ovarian carcinoma cell line OVCAR-3, which served as positive control (inset). B, strong cytoplasmic and focal membranous expression of CD24 in the HT29 colon cancer cell line.

CD24 immunohistochemistry and statistical analysis. As described in other organs, CD24 immunostainings showed two separable staining qualities in colon tissue, a membranous and a cytoplasmic immunoreactivity. Normal colon mucosa was virtually CD24 negative, although some cases exhibited a weak membranous staining at the brush border. In general, colorectal carcinomas showed higher levels of CD24 expression, often with a relatively abrupt up-regulation in the transition zone of normal mucosa to neoplastic epithelium (Fig. 2A and B). The distribution of immunoreactivity in the tumors was fairly homogeneous, which made the evaluation of the staining intensity straightforward (0, +, ++, +++), and allowed omittance to incorporate the percentage of positive tumor cells in an immunoreactivity score. Details are given in Tables 2 and 3; for membranous CD24 staining, 68.7% of tumors were positive and 17% were strongly positive. Higher rates of membranous CD24 immunoreactivity were associated with systemic metastasis (pM₁) but showed no significant correlation to other clinicopathologic variables (Table 2). For cytoplasmic CD24 staining, we found 84.4% of cases positive and 10.2% cases showing a strong expression (Table 3). Cytoplasmic CD24 immunoreactivity was significantly associated with higher tumor stages (Dukes and pT), nodal or systemic metastasis (pN and pM), and finally tumor grade.

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Fig. 2.

CD24 immunohistochemistry. A, transition of low-grade dysplastic colonic epithelium in colon carcinoma. Note abrupt onset of predominantly membranous expression of CD24. B, interface between colon mucosa and colon carcinoma. Note strong predominantly cytoplasmic overexpression of CD24 in the neoplastic glands of this case. C, colon cancer with minimal focal membranous expression of CD24 (arrows). D, colon cancer with strong membranous expression of CD24 without cytoplasmic positivity. E, neoplastic colonic glands with strong cytoplasmic CD24 expression. F, liver metastasis of a colon carcinoma from the study cohort. Note strong simultaneous cytoplasmic and membranous expression of CD24.

Survival analysis. Cases with a strong cytoplasmic CD24 positivity showed a significantly shortened mean survival time in comparison with the other patients (P = 0.0018; Fig. 3A). This was even more evident in the group of non-pM₁ patients (P = 0.0003; Fig. 3B). In contrast, no association of membranous CD24 expression with patient survival times was demonstrable (Table 4; Fig. 3C).

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Fig. 3.

Patient survival in dependence of CD24 expression pattern. A and B, Kaplan-Meier survival curves for patients grouped for cytoplasmic CD24 expression (A, whole study cohort; B, patients without distant metastasis). Note dramatically reduced survival time for those patients whose tumors showed strong cytoplasmic positivity. C, survival curves for patients grouped for membranous CD24 expression.

Other prognostic factors in univariate survival analysis were Dukes stage, WHO tumor stage, nodal status, state of distant metastasis, and tumor grade (Table 4). Tumor localization was only a significant survival factor when the cases were grouped as either rectal or nonrectal carcinomas (P = 0.01). This is due to the tendency towards lower stages in our subgroup of rectal carcinomas, which is an effect of the exclusion of advanced rectal carcinomas treated with neoadjuvant therapy before operation.

In the multivariate survival analysis of the group of patients without distant metastasis, cytoplasmic CD24 expression remained a significant independent prognostic factor (P = 0.012) with a relative risk of 3.7 (confidence interval, 1.3-10.3). Other independent prognostic factors in multivariate survival analysis were the extent of tumor spread to the regional lymph nodes and patient age (Table 5). The prognostic value of strong cytoplasmic CD24 expression failed significance if all patients, including pM₁ cases, were analyzed (data not shown).

Discussion

CD24 is a small, glycosylated, mucin-like glycosylphosphatidylinositol-linked cell surface protein that is expressed physiologically in the developing pancreas and brain (19, 20), in regenerating muscle (21), and in normal keratinocytes (22) and renal tubules (23). Its physiologic function is not fully elucidated, but it seems to be involved in control of cell proliferation, apoptosis, and cell adhesion. Overexpression of CD24 in lymphocytes suggested that CD24 levels can modulate the capacity of early T and B lymphoid progenitor cells to proliferate and survive. Antibody-based studies showed that cross-linking of CD24 induced apoptosis in B-cell precursors and suppressed the anti-CD40–induced proliferation of mature resting B lymphocytes (24). More recently, induction of apoptosis via CD24 was found in human Burkitt's lymphoma cells (25).

To date, the only CD24 ligand that has been identified is P-selectin. CD24 was able to support the adhesion of neutrophils or monocytes to activated endothelial cells or platelets, both of which express P-selectin. Additionally, this binding could be blocked by antibodies to P-selectin or CD24 (12). There is evidence suggesting a prometastatic role of CD24 in human tumor cells, which is based on this CD24-P-selectin interaction (13). Physiologically, P-selectin is expressed by activated endothelial cells and platelets and plays an important role in marginal adhesion and migration of cells under shear forces in the blood stream (12). Thus, it is conceivable that CD24-expressing tumor cells can disseminate more easily due to their capacity to form thrombi with activated platelets or to adhere to endothelia in the bloodstream, which has been showed for CD24-expressing breast cancer cells (15). In fact, we could show faster rates of disease progression of CD24-positive primary tumors for a variety of solid malignancies.

To our knowledge, this is the first comprehensive description of CD24 protein expression in colorectal carcinomas. In previous studies using the same methods, we have analyzed CD24 expression in epithelial ovarian cancer, breast cancer, non–small-cell lung cancer, prostate cancer, and pancreatic cancer (3–6, 26). With the exception of pancreatic cancer, we were able to show a significant association of higher CD24 levels in these tumors with earlier disease progression or earlier disease–related death. The definitions of CD24 expression rates and qualities to delineate low- and high-risk groups were slightly variable in these studies, but basically CD24-negative tumors were opposed to CD24-positive ones. Therefore, it is surprising to learn that in colorectal cancer, only the group with exceptionally strong cytoplasmic CD24 staining, which comprises 10% of cases in our study, suffered a markedly shortened survival, whereas the Kaplan-Meier curves for the other tumors did not differ significantly. Although we have noted in earlier studies that the cytoplasmic CD24 staining pattern is prognostically more significant than the membranous staining, the biological significance of cytoplasmic CD24 expression remains unclear. It could hint at an overproduction of CD24, at disturbances of CD24 distribution in the cell, or both. Very recently, Choi et al. (27) could reproduce our finding of cytoplasmic CD24 as a prognostic marker in epithelial ovarian cancer. More importantly, they investigated a larger cohort of ovarian borderline tumors and could clearly link cytoplasmic CD24 immunoreactivity to microinvasion. Apparently, the shift from apical membranous CD24 localization, which is observed in normal cells and in cells of very well differentiated glandular tumors, to the cytoplasmic CD24 localization reflects the transition of epithelial cells to a more invasive phenotype. This is reminiscent of the well-known epithelial-mesenchymal transition of tumor cells which is associated with the onset of invasiveness. In the cytoplasm, CD24 is stored in microvesicular bodies, which can be shed by tumor cells as exosomes into the environment and circulation.⁴

Linked to the cell membrane by a glycosylphosphatidylinositol anchor, CD24 is physiologically localized in lipid rafts, which represent small membrane microdomains serving as a platform for the regulation of cell adhesion and signaling (28). Within rafts, HSA/CD24 is closely associated with src kinases, G proteins, and calcium channels. Signal transduction via Tyr phosphorylation and src kinases has been described on triggering of many glycosylphosphatidylinositol-anchored molecules (29) but a specific signaling cascade for CD24 has not yet been identified, which ought to be the focus of further study.

The finding of cytoplasmic CD24 as an independent marker of high-risk patients is important, although the group of patients with strongly CD24-expressing colorectal cancers is comparatively small. This information could assist us in our attempt to individualize patient care (e.g., monitoring more closely the patients under concern for disease recurrence or progression after surgery) or even to consider an intensified adjuvant therapy.

In summary, we found CD24 overexpression in neoplastic colonic epithelia of carcinomas. Moreover, strong cytoplasmic CD24 expression was associated with shortened patient survival times in univariate and multivariate analyses, which clearly warrants further studies to clarify the biological role of CD24 in colorectal cancer.