In most cases, patients with clinically localized prostate cancer treated locally with modalities such as surgery or radiation therapy will be cured of their disease. However, a proportion of men will harbor microscopic localized or metastatic residual disease. These patients will ultimately go on to develop biochemical recurrence of disease and eventually are at risk of developing clinical metastatic progression and death from their prostate cancer. The risk of progression after local therapy is generally estimated based on available clinicopathologic variables. For example, after radical prostatectomy it is estimated based on both clinical data, such as pre-surgical PSA, as well as pathologic data such as extracapsular extension, seminal vesicle involvement, surgical margin status, and the Gleason score. These approaches described by Amico et al., Kattan et al., and others have improved our ability to risk stratify patients through a continuous variable taking in to account these types of variables with high specificity, yet relatively low sensitivity (21
). Due to the lower sensitivity of such available risk stratifying measures it has been difficult to justify the routine use of adjuvant therapy prior to frank biochemical recurrence. Several randomized phase III trials have tested the use of routine, adjuvant radiation therapy after prostatectomy in men with high-risk disease estimated using only clinicopathologic parameters (24
). These studies found significant improvement in biochemical recurrence-free survival in men with adjuvant radiation therapy, but no improvement in overall survival (27
). An inability to identify men at high risk may have contributed to the equivocal outcome. This was in no small part due to deficiencies in our ability to accurately risk-stratify patients using only clinicopathologic variables. With the addition of the chemokines identified in this study to more standard clinicopathologic variables we were able to predict the risk of recurrence among men who after prostatectomy with greater accuracy than by clinicopathologic parameters alone. The logistic regression model that included the three chemokines improved the specificity from 36% to 72% at 90% sensitivity, when compared to clinicopathologic parameters alone (). This corresponded with a significant improvement in the AUC for the model that included all three chemokines to 87.7% versus 80.6% for only clinicopathologic variables.
The chemokines identified through the lectin-based enrichment method have specific positive and negative biological roles in prostate cancer progression. All eukaryotic organisms glycosylate proteins exposed to the extracellular space. Since lectins specifically bind such glycosylation groups, we processed the prostatectomy specimens with wheat germ agglutinin resin in batch and were able to observe the expression of a number chemokines otherwise not detectible. Inflammatory cells are emerging as potential mediators of cancer metastasis (11
). Both CX3CL1 and CCL4 can recruit NK cells, T cells, and monocytes. Based on our data however, the two chemokines seem to reflect an opposite status of prostate cancer recurrence. Accordingly, apart from similar inflammatory recruitment characteristics, CCL4 has direct proliferative and migration effects on prostate cancer cells in vitro
). Conversely, CX3CL1 is reported to reduce migration of prostate cancer cells in culture (29
). Finally, IL-15 can prevent prostate cancer progression by supporting NK-cell function in vivo
). CX3CL1 and CCL4 overwhelmingly was supported prediction of prostate cancer biochemical recurrence under all univariant and multivariant criteria tested. Although the univariable analysis suggested IL-15 to be significant, as illustrated by Kaplan-Meir plot (), the overall multivariable models (logistic regression and Cox proportional hazard) did not indicate statistical significance in predicting recurrence-free survival ( and ). Nevertheless, specific multivariable one, three, and five-year survival analysis supported the importance of IL-15 as a predictive factor, illustrated in the nomogram (). The significant contribution of each of the chemokine biomarkers needs to be taken in context of clinicopathologic parameters. The well-recognized predictors for biochemical recurrence, like Gleason scores and seminal vesicle involvement, were greater in the population studied (), however their predictive significance was diminished when compared to the chemokine biomarkers in a multivariable analysis (, ). Together, the data argue for considering multiple biomarkers in the prediction of those prostate cancers likely to have the greatest clinical significance, especially when coupled to clinicopathologic parameters.
We have shown in this report that a cumulative evaluation of the expression by a continuos variable was able to accurately predict biochemical recurrence after radical prostatectomy. Using a model incorporating chemokines and clinicopathologic variables developed in this study, men could be identified who were at substantially higher risk of recurrence after prostatectomy. While local recurrence can potentially be treated with prostatic bed salvage radiation, systemic disease is treated with hormonal therapy, of which the exact timing is controversial. However, of the 82 subjects with biochemical recurrence, 44 (54%) showed elevated PSA within one year of surgery. These apparent early recurrence subjects likely had already developed local or distant metastasis prior to surgery. In an era where there is growing concern that many prostate cancers are over-treated, the use of this model could support the clinical determination of those prostate cancers that would progress to symptomatic disease and ultimately death, as well as potentially provide novel biologic targets to inhibit the metastatic progression. At the same time, it has the potential to identify those patients who harbor more indolent disease that could be managed by active surveillance and may be spared the potential morbidity of aggressive local therapy. In principal, the same model that was generated in this study could be done on diagnostic biopsy specimens and incorporated into a model utilizing clinical variables such as the PSA, clinical Gleason Score, and clinical stage to more accurately predict a patient’s disease risk prior to any therapy. It should be noted, however, that this idea was not formally tested in this study as the specimens were obtained on freshly removed prostates after prostatectomy. Nevertheless, the cores utilized here were of similar nature as would be obtained at the time of diagnostic biopsy of the prostate. Notably, many of the tissues processed had no evidence of adenocarcinoma in the sample (). As the biomarkers are secreted factors, it seems that actual tumor sampling is not particularly necessary. However, more studies are needed to know the extent of adjacency required for positive prediction ability. Therefore, the same techniques used in this study can be readily transferred to the clinical setting at the time of prostate biopsy. Previously reported blood or tissue biomarker analysis have not approached the sensitivity and specificity of the prediction achieved by the model described here (31
In summary, we have shown that prostate tissue levels of three chemokines: CCL4, CX3CL1, and IL-15, are predictive of biochemical recurrence in men who have undergone radical prostatectomy for adenocarcinoma of the prostate with high specificity and sensitivity. Further, we have shown that a nomogram that incorporates these three chemokines with other established clinicopathologic variables is a better predictor of outcome than using clinicopathologic variables alone. This suggests that CCL4, CX3CL1, and IL-15 are biomarkers of prostate cancer recurrence after radical prostatectomy.
Statement of Translational Relevance
Current therapeutic options for prostate cancer patients involve surgical or radiation based ablation of the prostate. While this can be curative for many patients, up to 35% of prostatectomy patients develop biochemical recurrence of prostate cancer. The high-risk prostate cancer patient population often dies from the disease due to metastatic progression. Early detection of prostate cancer likely to develop biochemical recurrence can lead to proactive use of adjuvant therapeutic options prior to frank biochemical recurrence. As tumor associated inflammatory changes can regulate prostate cancer metastatic progression, we looked to chemokines in prostatectomy tissue as potential indicators of future biochemical recurrence. We identified three chemokines that independently predict biochemical recurrent status within five years of prostatectomy in a nested case control study. The differentially expressed chemokines further supplemented known clinicopathologic variables to provide high sensitivity and specificity. The multivariable prediction models generated resulted in a nomogram that provides superior prediction capacity.