Quantitative real-time RT-PCR of CD24 mRNA in the detection of prostate cancer

Since 2003, prostate cancer has been the tumor with the highest incidence among men in the Federal Republic of Germany. Approximately 40,600 new cases and 11,100 deaths from this disease were reported for this year in Germany [1]. A precise pathological classification is of essential importance for treating both localized and advanced tumors. Established conventional prognostic markers are prostate-specific antigen (PSA) [2], the tumor stage and grade [3], the patient's age [4], and the presence of residual tumor(s) [5].

PSA shows high sensitivity but very low specificity as a serum marker for prostate cancer. This leads to an enormous increase of basically superfluous prostate biopsies and resultant high costs [2].

Although most of the initially diagnosed tumors are organ-confined (pT2 stage), they are still highly variable in their clinical outcome. This points to the shortcomings/limits of the classical prognostic markers and underscores the need for improved markers.

A comparison of benign, organ-confined, and highly advanced tumors using gene expression profiling techniques has recently disclosed several new molecular markers with prognostic value for prostate cancer [6–8]. A similar approach was used to unmask CD24 as a significant predictor of PSA relapse and poor prognosis in low-grade prostate cancer [9]. CD 24 is a small, heavily glycosylated, mucin-like cell surface protein [10]. It is expressed in granulocytes and various developing cells, including pre-B cells, keratinocytes, and renal tubular epithelium, but also in a large variety of malignancies [11–20]. CD24 is a ligand of P-selectin and could thus promote the dissemination of CD24-positive tumor cells [21].

We evaluated the expression of CD24 by quantitative fluorescence-based real-time RT-PCR (LightCycler) as an alternative to the predominant immunohistolological staining procedures (CD24-specific monoclonal antibodies).

Our aim was to determine whether QRT-PCR-based detection of CD24 will yield data with sensitivity and specificity comparable to those in existing reports based entirely on immunohistochemical staining. In this case, quantitative CD24 mRNA evaluation could supplement the pathologist's subjective analysis in doubtful cases.

The study thus examined 55 probes obtained in cases of benign prostatic hyperplasia (BPH) and 59 from patients with prostatic adenocarcinoma (Pca).

Graphs depict relative CD24 expression results in Figures 1 and 2.

The mean relative CD24 expression value was 0.21 (range 0.013–1.02; median 0.14) in the Pca group and 0.08 (range 0.01–0.29; median 0.06) in the BPH group. This Pca patient population had a 2.69 times higher mean CD24 expression for Pca than for BPH. This difference was highly significant (p < 0.0001). Analysis of the CD24-ROC curve (Figure 3) shows a moderate specificity (47.3%) but high sensitivity (86.4%) if the cutoff is set at 0.0498. The Area under the curve was 0.754. The positive predictive value was 64.9% (50/77 patients), whereas the negative predictive value was 75.7% (28/37 patients). Analysis of the ROC curve of the initial PSA yielded an area under the curve of 0.932 (Figure 4).

The Pca group showed no significant correlations between the relative CD24 expression and various clinical parameters, including age, clinical stage, initial PSA, pathological stage, lymph node metastases, margins, grading (WHO), and Gleason Score (Data not shown).

The follow-up (mean 3 years, range 2.4 – 3.3 years) disclosed biochemical progression in only two patients after radical prostatectomy. The relative CD24 expression was within the mean range of all Pca patients at 0.202 in the first case (pat. 70) and 0.42 in the second one (pat. 74). Two patients with palliative resections showed clinical progression. Their relative CD24 expressions were 0.3552 (pat. 102) and 0.075 (pat. 105). These values did not correspond to a significant correlation.

The discovery of prostate-specific antigens (PSA) in 1970 [22] revolutionized the diagnostics of prostatic adenocarcinoma. The good sensitivity but poor specificity of this serum marker has led, on the one hand, to a prognostically favorable stage shift towards increasingly smaller tumors and, on the other hand, to numerous basically superfluous prostate biopsies without tumor cells or with extremely small foci. This offers a great challenge to the assessing pathologists.

In recent years, a great number of new molecular markers (DNA, RNA, protein) for many tumors, including prostate cancer, have been discovered by microarray technologies [23–28]. The clinical value of these upcoming new markers must now be determined in larger clinical studies.

One of these markers is CD24, which was recently described by Kristiansen et al. based on RNA expression profiling. 102 tissue samples from the prostate and 31 samples from lymph node metastases were examined. The immunohistology used in their study achieved a sensitivity of only 48% in the prostate tissue samples and 68% in the lymph node samples. This involved strong staining in only 12% and 29% of the cases and only a very weak reaction in most of them [9] there was no association with tumor stage or grade [8, 9]. However, since no benign control tissue was examined in this study, there was no calculated specificity. Kristiansen recently published the results of an exemplary CD24 RT-PCR in several laser-microdissected prostate tissue samples. Here a positive reaction was likewise achieved in only 38.5% of the cases [8]. The high heterogeneity of CD24 expression even within a tumor of the same individual is apparently a problem not only for the CD24 immunohistochemistry of the prostate carcinoma but also for other tumor entities. The general problem of reproducibility of an immunohistochemistry is particularly accentuated by this [9].

Based on the RT-PCR protocol using LightCycler technology, the present study revealed a highly significant elevation of tissue CD24 mRNA expression in Pca patients compared to patients with a benign histology. The sensitivity (correctly detected cancer patients) found in our series (86.4%) is much better than that reported in other series (38.5% and 48% [8, 9]). The minimal tumor component yielding a positive result is still unclear. If CD24 mRNA should be detectable not only in tissue but also in prostatic secretions or even urine, the RT-PCR presented here could serve as an important noninvasive supplement to other procedures (digital rectal examination and serum PSA) within the framework of cancer screening. We are currently performing examinations in this connection. However, the moderate specificity (correctly detected patients without cancer) of the technique presented here currently prevents this test from replacing or even supplementing other pathological procedures.

The histology of all patients was known in advance. All BPH 55 patients had either a low preoperative PSA or a previous biopsy to exclude malignancy. Thus we are dealing with a PSA selection. This is corroborated by the far-above-average AUC of the initial PSA (0.932) depicted in Figure 4. Thus, a combination of the predictive values of PSA and CD24-mRNA is not useful, at least not on the basis of the present data.

Laser microdissection was primarily dispensed with, since we wished to streamline the procedure, and Kristiansen et al. have already described the heterogeneity of CD24 expression. We therefore analyzed tissue previously diagnosed for more than 50% tumor infiltration by an uropathologist (J.K.).

The manufacturer designates PBGD as an ideal reference and housekeeping gene and points out that this statement is based on extensive internal series of experiments. The PBGD expression level remained stable with widely varying experimental setups and was also within the RNA expression range of the target gene. No other reference genes were used.

Other study groups found the following results for other tumor entities: Fogel shows that CD24 was expressed in four of six breast cell lines (66.7% sensitivity) [11]. Droz demonstrated that clear cell renal carcinomas always show a strong reaction (100% sensitivity) [19]. The sensitivity was 84% [29] for ovarian carcinomas and 76% [30] for non-small-cell lung carcinomas. Huang showed that CD24 mRNA is expressed in 66% of hepatocellular carcinomas (HCC) [12]. None of the above-mentioned studies examined malignant compared to benign tissue. Thus only conjectures can be made regarding the specificity of the procedures.

We also found no correlation between CD24 expression and clinical or pathological parameters like the tumor stage or metastatic spread. Even specimens from patients with early recurrences did not differ significantly from those obtained in other stages of the disease. Only two of 13 Pca patients with a relative CD24 expression of over 0.3 are currently in disease progression (as indicated by a PSA rise or clinical progression).

Our findings are entirely at variance with results previously described by Kristiansen et al., which led them to assess CD24 as a strong independent predictor of recurrence not only for Pca but also for lung, breast, and ovarian cancer [9, 29–31]. Additional multicenter follow-up studies are necessary to confirm the value of CD24 RT-PCR as a prognostic marker.

Normalized CD24 transcript levels in 59 Pca cases were increased 2.69-fold on the average when compared to 55 BPH cases. This difference was highly significant (p < 0.0001).

The procedure has a very good sensitivity (86.4%) and moderate specificity (47.3%).

CD24 expression levels in Pca cases were not statistically associated with the tumor or lymph node stage or with grading (WHO), surgical margins, or the Gleason score.

The present study demonstrates the feasibility of quantitative CD24 RNA transcript detection in prostatic tissues even without previous laser microdissection. However, the results do not yet signify a major improvement over the usual standard tissue diagnostics.

If CD24 mRNA should be detectable not only in tissue but also in prostatic secretions or even urine, the RT-PCR presented here could serve as an important noninvasive supplement to other procedures (digital rectal examination and serum PSA) within the framework of cancer screening.