Preferential Expression of Chemokine Receptor CXCR4 by Highly Malignant Human Gliomas and Its Association with Poor Patient Survival (2008)

To the Editor: We read with great interest the recent report by Bian et al. (4) concerning their investigation of CXCR4 expression in malignant gliomas and its correlation with patient prognosis. CXCR4 is a cell membrane-spanning G-protein-coupled chemokine receptor with pleiotropic functionality governing cellular migration and chemotaxis in multiple organ systems. The pathophysiological role of the CXCR4 signaling pathway in cancer biology has received increasing attention over the past decade (2). In the specific context of gliomas, prior work by our group and others has demonstrated that the expression of CXCR4, and its sole protein ligand CXCL12, mediates tumor growth (3,11), invasion (5), and angiogenesis (1). Recent data from our laboratory (M Ehtesham, unpublished data) also indicate that the CXCR4-CXCL12 axis may serve as a key regulator of cancer stem cell turnover within glioblastoma multiforme (GBM). As such, we welcome the data of Bian et al., which serve to further corroborate the important role that CXCR4 signaling plays in glioma biology and underscore the potential therapeutic utility of targeting this pathway in patients with GBM. Importantly, their demonstration that the lipoxygenase inhibitor Nordy could decrease CXCR4 expression and impair tumorigenicity in U87 xenografts in nude mice represents a novel treatment approach that is deserving of further investigation. There are, however, several aspects of the report by Bian et al. regarding which we would like to express certain concerns. These relate chiefly to their claim, as stated in the article title, that CXCR4 expression correlates with patient prognosis. As is the case in many cancers, and particularly with GBM, the search for molecular markers that may be used as surrogate tools to predict clinical outcome remains a critical research objective. It is logical that ideal prognostic markers would consist of proteins that functionally govern key aspects of a tumor's biology, and, therefore, CXCR4 certainly represents a promising target. Indeed, expression of CXCR4 has already been correlated with poor prognoses in patients with osteosarcoma (8), melanoma (6), and colon cancer (7). We have speculated for some time that CXCR4 may provide similar utility in the setting of GBM and were, therefore, very interested in evaluating the data in the article by Bian et al. We are concerned, however, that the clinical data sets used to support their conclusion that CXCR4 expression levels relate to patient outcome have not been evaluated with the rigor required for such a claim to be forwarded. First, their patient grouping methodology pools together Grade III and GBM tumors into a single population. They then divide this population into patients who survived for more than 3 years and those who succumbed to disease within 3 years. This is done without providing any data relating to what proportion of patients surviving for more than 3 years had Grade III tumors as opposed to GBMs. This is a critical confounding factor, as it is already known that GBMs express higher levels of CXCR4 than Grade III tumors (10), and since Bian et al. do not provide a differential breakdown of their survival populations, one might speculate that the CXCR4-low patients who end up surviving for more than 3 years are likely to overwhelmingly consist of patients with Grade III gliomas, whereas the CXCR4-high cases succumbing within 3 years are predominantly GBM patients. If this is the case, their data may potentially have been misinterpreted as having prognostic significance when none actually exists. Furthermore, the authors themselves state that, in their sample set, CXCR4-high tumors were larger. They do not provide an explanation as to how this key difference in average tumor size (as found in their data analysis) between CXCR4-high and CXCR4-low patients was controlled for and how they were able to conclude that CXCR4-high patients had a worse prognosis specifically related to CXCR4 expression level and not simply because of having a larger disease burden. For more accurate evaluation, their data sets should first be controlled for fundamental clinical variables including, but not necessarily limited to, patient age and tumor size. Furthermore, patients should be divided into discrete categories of Grade III versus GBM for independent evaluation of the correlation between CXCR4 expression level and clinical outcome within each of these tumor grades. Second, there is no detail provided regarding the clinical treatment course that these patients were subjected to and how variability in therapeutic protocols from patient to patient was controlled for. Important variables such as duration and nature of radio- and chemotherapeutic treatment as well as essential details as to whether individual patients underwent image-complete or subtotal operative resections are not discussed. All of these parameters can markedly affect clinical outcome, and, in the absence of these data, it is very difficult to evaluate what correlation CXCR4 expression may directly have with patient prognosis. Furthermore, the authors present only a cursory description of the generalized statistical methodology they used for their experiments. A significantly more detailed outlay of the statistical treatise they used in the evaluation of their clinical data sets would be very welcome. Third, Bian et al. use a semiquantitative methodology for the evaluation of CXCR4 expression in their clinical samples. This consists of immunohistochemically staining tumor tissue samples and then categorizing samples into three separate groups based on the percentage of cells within the observed microscopic fields that are labeled as positive. This is potentially misleading, as the immunohistochemical methodology used is based on serial amplification of signal strength to facilitate visual detection of antibody-antigen binding. Although this serves as an excellent tool for detection of low-expression antigens within tissue samples, it may provide misleading results if analyzed for quantitative purposes. The field of oncology is moving toward more quantitative expression analyses, and techniques such as fully quantitative real-time polymerase chain reaction have supplanted immunohistochemistry and gel-based densitometry as the “gold standard” for detailing differential expression levels across multiple patient samples. In summary, we feel that Bian et al. have added to the body of literature detailing the involvement of CXCR4 in glioma biology. Their conclusions relating to patient prognosis, however, are difficult to evaluate in light of the limitations we have described. Given the importance of finding prognostically significant molecular marker(s) for patients with high-grade gliomas, and the biological rationale supporting the candidacy of CXCR4 as a viable platform for this purpose, we would welcome a more rigorous evaluation than that presented. Moneeb Ehtesham Charles B. Stevenson Reid C. Thompson Nashville, Tennessee