We determined the immunodulatory effects following a brief exposure of anti-CTLA-4 in patients with urothelial carcinoma of the bladder requiring surgery. This study, to our knowledge, is the first analysis of CTLA-4 blockade in the setting of urothelial carcinoma and in the setting of a pre-surgical clinical trial. The study population consisted of 12 patients, with 6 patients receiving 3mg/kg/dose of anti-CTLA-4 and another 6 patients receiving 10mg/kg/dose for 2 doses prior to surgery. The treatment was found to be tolerable in our cohort of patients with 11/12 patients receiving both doses of antibody. Grade 1-2 diarrhea and rash were the most common drug-related side effects. Of relevance, the only noted grade 3 irAEs were ischemic papillopathy and diarrhea, which were both responsive to treatment with steroids. Patients have been followed for a median of 20 months with 9/12 patients continuing to remain without evidence of disease. In addition, the pre-operative approach in this study overcomes a major hindrance of biological studies, in that it provides for analyses of tumor tissues. The availability of cystectomy specimens provided sufficient tissue to perform detailed immunological analyses, which could be correlated with biomarkers in peripheral blood.
Data from our preliminary results on the first 6 patients treated with 3mg/kg/dose of anti-CTLA-4, as well as the current analyses from the 6 patients treated at 10mg/kg/dose demonstrated that treatment with anti-CTLA-4 antibody leads to an increase in the frequency of CD4+ICOShi cells, which comprise a population of effector T cells. This population of CD4+ICOShi cells, which was found to be increased in both tumor tissues and peripheral blood, represented a potential biomarker that could be used for correlation with clinical outcome in patients with metastatic disease who receive treatment with anti-CTLA-4. A retrospective analyses of peripheral blood samples obtained from patients with metastatic melanoma who were treated with anti-CTLA-4 antibody at 10mg/kg/dose demonstrated that a sustained increase in CD4+ICOShi T cells at week 12, after 4 doses of therapy, correlated with improved overall survival.
Our clinical trial does have limitations. The analyses of untreated bladder tumor tissues were performed on samples obtained from a separate group of patients since cystectomy samples consisting of the entire bladder were necessary in order to obtain sufficient numbers of cells for immunological analyses. Therefore, specimens from stage matched untreated patients were utilized for comparison in flow cytometry studies. This does allow for confounders in that we could not account for unknown factors that may impact our data. The general trend in the group of patients suggests that although there may be unknown factors that could account for differences between patients in the two separate groups, the increased frequency of CD4+ICOShi cells after treatment serves as a marker of CTLA-4 blockade that supersedes minor contributions from unknown variables. Another limitation is that our small sample size limits the statistical power of the immunological correlates. More patients will need to be analyzed in larger studies.
In our current study, we report that anti-CTLA-4 is tolerable in the pre-operative setting, which provides an opportunity to obtain immunological data from both tumor tissues and the peripheral blood. Our data, consisting of an increased frequency of CD4+
T cells, were obtained in a cohort of patients with localized urothelial carcinoma of the bladder but nonetheless may be relevant in patients with other types and stages of malignancies, such as metastatic melanoma, as we report here. Our data support the concept that a sustained increased frequency of CD4+
T cells may serve as a biomarker of anti-CTLA-4 activity and/or as a clinical benefit for patients who are being treated with this novel agent. We have shown that anti-CTLA-4 can induce similar immunological changes in patients with different types of malignancies and in different tumor tissues (25
). Future studies with additional patients will need to be conducted in order to validate CD4+
T cells as a biomarker to optimize anti-CTLA-4 therapy and/or indicate potential clinical benefit thus warranting continued therapy.
Ours are the first studies to demonstrate alterations in ICOS expression on T cells during anti-tumor responses. It is of considerable interest to determine the role of ICOS and its interactions with its ligand in these responses. ICOS is expressed on both effector and regulatory T cells and has a very complex biology, having been variously implicated in T/B cell interactions, germinal center formation, and regulation of Th1 and Th2 cytokines (26
). A key fact may be that it has the potential to signal via the PI3 kinase pathway and may enhance T cell survival. Mechanistic studies are ongoing to understand how the ICOS pathway affects anti-tumor responses.
In summary, we have shown that anti-CTLA-4 can be delivered in the pre-operative setting without an increase in surgical complication. The previously reported toxicities associated with anti-CTLA 4 therapy were observed and appropriately managed. Furthermore, the pre-operative model can serve as a powerful discovery platform to efficiently identify biomarkers that can be applied to the metastatic disease setting to establish the relevance of experimental findings. Our identification of ICOS is the first immunologic marker to be identified in both tumor tissues and the peripheral blood of patients treated with anti-CTLA-4 thus providing a relevant biomarker on which to build future immune monitoring strategies.